Methods and compositions for risk stratification

ABSTRACT

The present invention provides an approach for the simultaneous determination of the activation states of a plurality of proteins in single cells. This approach permits the rapid detection of heterogeneity in a complex cell population based on activation states, and the identification of cellular subsets that exhibit correlated changes in activation within the cell population. Moreover, this approach allows the correlation of cellular activities or properties. In addition, the use of potentiators of cellular activation allows for characterization of such pathways and cell populations.

This application is a continuation of U.S. Ser. No. 12/778,847, filed onMay 12, 2010, currently pending, which is a continuation of U.S. Ser.No. 12/125,759, filed on May 22, 2008, currently pending, which is acontinuation of U.S. Ser. No. 10/898,734, filed on Jul. 21, 2004, nowU.S. Pat. 7,393,656, all of which are hereby expressly incorporated byreference in their entirety.

FIELD OF THE INVENTION

The invention relates generally to the field of protein detection usingflow cytometry. More specifically, the invention relates to usingpolychromatic flow cytometry to identify signal transduction events andcellular activation profiles. The invention further relates to the useof potentiators that impact activation states and thus allow for furthercharacterization of signal transduction events and activation profiles.

BACKGROUND OF THE INVENTION

Proteins are major components of cells and the spatiotemporal expressionpattern and the subcellular localization of proteins determine theshape, structure, and function of cells. Many proteins are dynamicallyregulated so that their activity is altered in response to certainintrinsic or extrinsic cues frequently referred to herein as “activationevents”. Such regulation generally occurs in the context ofprotein-protein interaction networks commonly described as signaltransduction cascades. The individual members of such cascades oftenexist in either active or non-active states, and it is the conversionbetween these states that leads to propagation or inhibition of a signalthrough the cascade. Given their integral role in cellular developmentand function, dysregulation of such cascades can lead to numerousdiseases, and in particular those diseases involving improper cellproliferation such as cancer.

Researchers investigating signal transduction cascades havetraditionally used methods that rely on lysates of cellular populations,e.g. western blots. Such methods have a number of inherent limitationsthat can obscure key information. For example, using populations ofcells to produce a lysate hightens the possibility that rare cell eventswill be diluted to an extent that key activation events are no longerevident. In addition, the use of lysates can only approximate the nativecellular activation profiles of signal cascades, as lysis inherentlyalters the cascade environment. Given such limitations, new methodscapable of focusing on the individual members of signal transductioncascades in native environments are necessary to better understand therole of activation events in disease.

Accordingly, the present invention provides an approach for thesimultaneous determination of the activation states of a plurality ofproteins in single cells. This approach permits the rapid detection ofheterogeneity in a complex cell population based on activation states,and the identification of cellular subsets that exhibit correlatedchanges in activation within the cell population. Moreover, thisapproach allows the correlation of cellular activities or properties,including the ability to classify disease states based on signaltransduction activation state. In addition, the use of potentiators ofcellular activation allows for further characterization of such pathwaysand cell populations.

SUMMARY OF THE INVENTION

In a preferred embodiment, the instant invention provides a method fordetecting the activation state of at least two activatable proteins ineach cell of a cell population. The first step of this method is toprovide a population of cells that have the two activatable proteins.That step is followed by contacting the population of cells with aplurality of activation state-specific binding elements. Theseactivation state-specific binding elements have at least a firstactivation state-specific binding element that binds to a first isoformof the first activatable protein; and a second activation state-specificbinding element that binds to a first isoform of the second activatableprotein. The presence or absence of binding of the elements is thendetected using flow cytometry to determine the activation state of thetwo proteins.

In an additional embodiment, the two binding elements are labeled withdifferent labels.

In an additional embodiment, the cells tested are primary cells.

In an additional embodiment, the activation state information of thefirst and second activatable proteins is used to determine the cellularstatus of at least one cell.

In an additional embodiment, the cellular status determined is drugsusceptibility.

In an additional embodiment, the population of cells is contacted with acandidate agent prior to being contacted with the binding elements.

In an additional embodiment, the candidate agent is a potentiator.

In an additional embodiment, the potentiator is a cytokine.

In an additional embodiment, the potentiator is a growth factor.

In an additional embodiment, the activation state of at least one of theactivatable proteins is a phosphorylation state.

In an additional embodiment, at least one of the activatable proteins isa kinase.

In an additional embodiment, at least one of the activatable proteins isa caspase.

In an alternative embodiment, the instant invention provides a methodfor detecting the activation state of at least one activatable proteinin each cell of a population of cells. The first step of the method isto provide a population of cells comprising the first activatableprotein. The next step involves contacting the population of cells witha plurality of substrates, where the plurality of substrates includes atleast one substrate that is modified by the activatable protein. Flowcytometry can then be used to detect the activation state of theactivatable protein by measuring the amount of substrate that has beenmodified.

In additional embodiments, the present invention provides methods andcompositions for simultaneously detecting the activation state of aplurality of activatable proteins in single cells using flow cytometry.The invention further provides methods and compositions of screening forbioactive agents capable of coordinately modulating the activity oractivation state of a plurality of activatable proteins in single cells.The methods and compositions can be used to determine the proteinactivation profile of a cell for predicting or diagnosing a diseasestate, and for monitoring treatment of a disease state. Further, themethods and compositions of the present invention can be used optionallyto sequentially detect the activation state of a plurality ofactivatable proteins in single cells. In addition, the methods andcompositions of the present invention can be used optionally detect theactivation state of a single protein or modulate the activity oractivation state of a single protein.

The invention provides populations of cells, single cells, cell lysates,proteins, and samples comprising populations of cells, single cells,cell lysates, proteins useful in the methods of the present invention.In particular, the invention provides activatable proteins andactivation state-specific antibodies that bind to a specific isoform ofan activatable protein. In one aspect, the activation state-specificantibodies are conjugated to a label, preferably a fluorescent label,and more preferably a FRET label.

In one aspect, the invention provides methods of detecting theactivation state of at least a first and a second activatable protein insingle cells, the method comprising the steps of: a) providing apopulation of cells comprising the first and the second activatableproteins; b) contacting the population of cells with a plurality ofactivation state-specific antibodies, wherein the plurality ofactivation state-specific antibodies comprise: i) at least one firstactivation state-specific antibody that is capable of binding to acorresponding isoform of the first activatable protein in the populationof cells; and ii) at least one second activation state-specific antibodythat is capable of binding to a corresponding isoform of the secondactivatable protein in the population of cells; and c) using flowcytometry to detect the binding of the first and second activationstate-specific antibodies in single cells of the population of cells,wherein the binding of the first activation state-specific antibody isindicative of a specific activation state of the first activatableprotein, and the binding of the second activation state-specificantibody is indicative of a specific activation state of the secondactivatable protein.

In a further aspect, the first activatable protein is a kinase. Also ina further aspect, the first activatable protein is a caspase.

In a further aspect, the first activatable protein is a first kinase andthe second activatable protein is a second kinase. Also in a furtheraspect, the isoform of the first kinase is a first phosphorylatedkinase, and the isoform of the second kinase is a second phosphorylatedkinase. In another aspect, the first activation site-specific antibodybinds to the first phosphorylated kinase, and the second activationsite-specific antibody binds the second phorphorylated kinase.

In a further aspect, the first activatable protein is a first caspaseand the second activatable protein is a second caspase. Also in afurther aspect, the isoform of the first caspase is a cleaved product ofa first pro-caspase, and the isoform of the second caspase is a cleavedproduct of a second pro-caspase. In another aspect, the plurality ofactivation site-specific antibodies comprise a first activationsite-specific antibody that binds to the isoform of the first caspase,and a second activation site-specific antibody that binds to the isoformof the second caspase.

In another aspect, the invention provides methods of detecting theactivation state of at least a first activatable protein in singlecells, the method comprising the steps of: a) providing a population ofcells comprising at least the first activatable protein; b) contactingthe population of cells with a plurality of substrates; wherein theplurality of substrates comprise at least a first substrate that iscapable of being modified by a corresponding isoform of the firstactivatable protein in the population of cells; and c) using flowcytometry to detect the modification of the first substrate in singlecells of the population of cells, wherein the modification is indicativeof a specific activation state of the first activatable protein.

In a further aspect, the population of cells further comprises a secondactivatable protein; the plurality of substrates further comprise asecond substrate that is capable of being modified by a correspondingisoform of the second activatable protein in the population of cells;and step c) further comprises using the flow cytometry to detect themodification of the second substrate in single cells of the populationof cells, wherein the modification of the second substrate is indicativeof a specific activation state of the second activatable protein.

In another aspect, the invention provides methods of detecting a proteinactivation state profile of single cells based on the activation stateof at least a first activatable protein in the cells, the methodcomprising the steps of: a) providing a population of cells comprisingat least the first activatable protein; b) contacting the population ofcells with a plurality of substrates; wherein the plurality ofsubstrates comprise at least a first substrate that is capable of beingmodified by a corresponding isoform of the first activatable protein inthe population of cells; c) contacting the population of cells with aplurality of activation state-specific antibodies, wherein theactivation state-specific antibodies comprise at least one firstactivation state-specific antibody that is capable of binding to acorresponding isoform of the first activatable protein in the populationof cells; and d) using flow cytometry to simultaneously detect: i) thebinding of the first activation state-specific antibody in single cellsof the population of cells, wherein the binding of the first activationstate-specific antibody is indicative of a specific activation, state ofthe first activatable protein; and ii) the modification of the firstsubstrate the single cells, wherein the modification is indicative ofthe specific activation state of the first activatable protein.

In a further aspect, the population of cells further comprises a secondactivatable protein; the plurality of substrates further comprises asecond substrate that is capable of being modified by a correspondingisoform of the second activatable protein in the population of cells;and step d) further comprises using the flow cytometry to detect themodification of the second substrate in the single cells, wherein themodification is indicative of a specific activation state of the secondactivatable protein.

In a further aspect, the plurality of activation state-specificantibodies further comprises a second activation state-specific antibodythat is capable of binding to a corresponding isoform of the secondactivatable protein in the population of cells, and step c) furthercomprises using the flow cytometry to detect the binding of the secondactivation state-specific antibody the single cells of the population ofcells, wherein the binding of the second activation state-specificantibody is indicative of the specific activation state of the secondactivatable protein.

In another aspect, the invention provides methods of screening for abioactive agent capable of modulating the activity of at least a firstactivatable protein in cells, the method comprising: a) providing apopulation of cells, each of the cells comprising at least the a firstactivatable protein, a second activatable protein, and a thirdactivatable protein, wherein the first activatable protein can activatethe second activatable protein thereby forming a specific isoform of thesecond activatable protein (isoform-2), and wherein the secondactivatable protein can activate the third activatable protein therebyforming a specific isoform of the third activatable protein (isoform-3);b) contacting the cells with a second activation state-specificantibody, a third activation state-specific antibody, and a candidatebioactive agent, wherein the second activation state-specific antibodyis capable of binding to the isoform-2, and wherein the third activationstate-specific antibody is capable of binding to the isoform-3; c) usingfluorescent activated cell sorting (FACS) to sort single cells of thepopulation of cells based on the presence of the isoform-2 and theisoform-3; and d) determining the ratio of the isoform-2 to theisoform-3 in the single cells in the presence and absence of thecandidate bioactive agent, wherein a difference in the ratio of theisoform-2 to the isoform-3 in the presence and absence of the candidatebioactive agent is indicative of the ability of the candidate bioactiveagent to modify the activity of the first activatable protein.

In a further aspect, the first activatable protein is activated by anactivating agent.

In a further aspect, the first activatable protein is a caspase. Inanother aspect, the first activatable protein is a kinase; in anadditional aspect, the kinase is PI3K; and in a further aspect the PI3Kis activated by a growth factor or by activation of a cell surfacereceptor.

In a further aspect, the kinase is PI3K; the second activatable proteinis PIP2; the third activatable protein is PIP3; the isoform-2 is PIP 4,5bisphosphate; and the isoform-3 is PIP 3,4,5.

In a further aspect, the first activatable protein is ICAM-2; theactivity is apoptosis; the second activatable protein is PIP2; the thirdactivatable protein is PIP3; the isoform-2 is PIP 4,5 bisphosphate; andthe isoform-3 is PIP 3,4,5. In a further aspect step a) furthercomprises clustering the ICAM-2, ICAM-3, or ICAM-1.

In a further aspect, the plurality of activation state-specificantibodies comprise at least one antibody selected from a group ofantibodies consisting of: anti-AKT-phospho-Ser473, anti-AKTphospho-Thr308, anti-p44/42 MAPK phospho-Thr202/Tyr204, anti-TYK2phospho-Tyr1054/1055, anti-p38 MAPK phospho-Thr180/Tyr182, anti-JNK/SAPKphospho-Thr183/Tyr185, anti-phospho-tyrosine, anti-phospho-threonine,anti-PIP2, and anti-PIP3.

In a further aspect, step a) further comprises contacting the populationof cells with an agent that induces the activation of at least the firstactivatable protein.

In a further aspect, step a) further comprises contacting the populationof cells with an agent that induces the activation of at least one ofthe first and the second activatable proteins.

In a further aspect, step a) further comprises contacting the populationof cells with an agent that induces the activation of at least one ofthe first, the second, and the third activatable proteins.

In a further aspect, the methods further comprise sorting the singlecells based on the activation state of the first activatable protein,and the activation state of the second activatable protein.

In a further aspect, the first activation state-specific antibodycomprises a first label, wherein the second activation state-specificantibody comprises a second fluorescent label and, wherein the sortingis by fluorescent activated cell sorting (FACS).

In a further aspect, the first activation state-specific antibodycomprises a first FRET label; the second activation state-specificantibody comprises a second FRET label and the sorting is by fluorescentactivated cell sorting (FACS).

In a further aspect, the first activation state-specific antibodycomprises a FRET label; the second activation state-specific antibodycomprises a label; and the sorting is by fluorescent activated cellsorting (FACS).

In a further aspect, step a) further comprises fixing the cells.

In a further aspect, the cells are mammalian cells.

In an alternative embodiment, the instant invention provides methods ofprognosing a clinical outcome for a patient that include first creatinga cytokine response panel representing a statistically sufficient numberof individuals, followed by creating a cytokine response panel for thepatient; and then comparing said patient's cytokine response panel tothe cytokine response panel representing a statistically sufficientnumber of individuals in order to come to a prognosis of the patient'sclinical outcome.

In an additional embodiment, the cytokine response panels are created byfirst detecting the phosphorylation state of multiple phospho-proteinsin non-stimulated cells then detecting the phosphorylation state ofmultiple phospho-proteins in cytokine-stimulated cells and determiningthe difference, if any, of the phosphorylation state of thephospho-proteins in the stimulated and non-stimulated cells.

In an additional embodiment, the detection is accomplished byintracellular phospho-specific flow cytometry.

In an additional embodiment, the phospho-proteins are selected from thegroup consisting of: Stat1, Stat3, Stat5, Stat6, p38, and Erk1/2.

In an additional embodiment, the cytokine is selected from the groupconsisting of: Flt3 ligand, GM-CSF, G-CSF or IL-3.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

A cytokine response panel reveals potentiated signal transduction nodesin primary acute myeloid leukemias. a, To survey the signal transductionnetwork of normal and leukemic cells a grid of six stimulationconditions by six phospho-protein targets was designed. Stimulationstates, shown in rows, included unstimulated or 20 ng/mL of FL, GM-CSF,G-CSF, IL-3, or IFN-γ. Target phosphorylations were detected usingphospho-specific antibodies for Stat1, Stat3, Stat5, Stat6, p38, andErk1/2, shown in columns. Each square in the grid represents theresponse of one phosphorylation site to one condition (termed the stateof a signaling node). The relationship between the grid and the flowcytometry data on which it is based is diagrammed for U937 cells. Inthese cells, the median fluorescence intensity (MFI) that showed thelargest increase compared to the unstimulated state was observed forphosphorylation of Stat1 following IFN-γ. b, Representative cytokineresponse panels of the HL-60 AML cell line, normal CD33+ leukocytes, andsix AML patient samples are shown. Repeat experiments using these AMLblasts yielded similar results (n=3), and variation among normal,healthy donors was minimal (n=6). The response to stimulation at eachsignaling node is calculated as log 2 (MFI stimulated/MFI unstimulated).

FIG. 2

Basal and potentiated signaling nodes that varied among tumor sampleswere used to define an AML biosignature. a, The cytokine response panelwas expanded to 30 total AML patient samples and rearranged to highlightthe basal and potentiated signaling of each phospho-protein. The firstrow of each has been colored to show the variation in the basalphosphorylation (relative to the minimum among the AML blasts). Of 900cytokine responses assayed, 93 (10.3%) displayed a detectablephosphorylation increase following stimulation (greater than 0.55 fold).The variance of node states was used to determine significance. b,Significant cytokine responses were restricted primarily to the 7/30cytokine response nodes with a variance across tumors greater than 0.1(yellow circles). c, A graph of the absolute median plotted against thevariance for each node state indicates the signal to noise threshold.The seven significant cytokine response states and the six basalphosphorylation states were used as a 13-parameter profile of tumorsignal transduction and were termed a biosignature.

FIG. 3

AML patients grouped by signal transduction biosignature form fourgroups that exhibit significant correlations to clinical prognosticmarkers. a, The 13-parameter biosignatures of differentiated CD33+myeloid cells from six normal blood donors (D01-D06), U937 and HL-60tumor cell lines, and 30 AML patient samples were grouped according tosimilarity using hierarchical clustering. The heat map for AML and tumorcell line cytokine responses was scaled to the donor sample medians toprovide a dynamic color range. Normal donor samples clustered togetherand are represented as a group. As shown previously, basal responses arerelative to the minimum among AML samples. b, Four main groups of AMLpatients were identified based on the similarity of their signaltransduction biosignatures. We designated these groups with SignalingCluster (SC) nomenclature based on the signaling which defined them andmapped several clinical markers within the identified patient groups.The patient samples with SC-P2 or SC-NP profiles displayed contrastingclinical markers and these markers frequently correlated with thecytokine responses defining the SC (significance assessed using χ2tests). SC-P2 and SC-NP represented 33.3% and 36.7% of patients,respectively. No patients with a SC-P2 profile who were treated withchemotherapy showed remission (9/9, p=0.002). The SC-P2 patient samplesfrequently had mutations in Flt3 (8/10), while patient samples with anSC-NP profile displayed few potentiated cytokine responses and nearlyalways expressed wild type Flt3 (9/11) (p=0.02). Samples with an SC-P2profile rarely expressed CD15/Lewis X antigen (9/10, p=0.03).Cytogenetic alterations were divided among branches of SC-P2 and SC-NP(20/20, p<0.001).

FIGS. 4A, 4B, 4C, 4D, 4E and 4F

Flt3 mutations in primary AMLs may potentiate myeloid signaltransduction nodes. FIG. 4A, Basal and cytokine response node states ofpatient samples with wild type or mutant Flt3 are shown for all 30 AMLsassayed. The MFI of phosphorylated Stat5 differed little in patientswith wild type Flt3 and patients with mutant Flt3. In contrast, GM-CSFinduced Stat5 phosphorylation (FIG. 4B) and G-CSF induced Stat5 (FIG.4C) and Stat3 (FIG. 4E) phosphorylation all correlated with Flt3mutation (p=0.04, 0.02, and 0.01 respectively). As a way of representingthe overall effect of Flt3 mutation on myeloid signaling we summed thecytokine responses of four myeloid signaling states from thebiosignature: p-Stat3/G-CSF (FIG. 4E) and p-Stat5/GM-CSF/G-CSF/IL-3(FIGS. 4B, 4C, and 4D, respectively). This summed myeloidcytokine response was significantly higher in AML patients with Flt3mutations (p=0.005; FIG. 4F).

FIGS. 5A and 5B

Representative 2D flow cytometry plots of Stat5 and Stat3phosphorylation following G-CSF stimulation in AML patient samples withwild type Flt3 (FIG. 5A) and mutant Flt3 (ITD) (FIG. 5B). 2D contourplot representations of Stat5 and Stat3 phosphorylation (y and x axisrespectively) in patient samples from SC-NP and SC-P2. Both the level ofbasal phosphorylation and the response to G-CSF are shown.

FIGS. 6A, 6B-1 and 6B-2

Tumor biosignatures and a potentiated model of tumor cell signaling.FIG. 6A, A general method for identifying a tumor biosignature is shownusing an example of STAT and Ras/MAPK signalling node states in AML.FIGS. 6B-1 and 6B-2, Composite maps of tumor networks from profilesSC-NP and SC-P2 were built out of common signaling events observed ineach cluster. Highlighted nodes were detected to be high basal orpotentiated in most of the samples from a profile group.

FIGS. 7A, 7B and 7C

Three AML patients profiled as SC-P2 showed similarities and differencesin potentiated signaling mechanisms. Pathway maps summarizing thesignaling phenotype of individual patient samples are shown for threeprofiled as SC-P2, as per FIG. 6B-1.

FIGS. 1-39, and the corresponding figure legends, of U.S. patentapplication Ser. No. 10/193,462 are hereby incorporated by reference intheir entirety.

DETAILED DESCRIPTION OF THE INVENTION

Intracellular assays of signaling cascades have been limited by aninability to measure and correlate functional data based on theactivation state of elements within specific signal transductioncascades. Such measurements and correlations are important fordistinguishing changes in signaling status arising in rare cell subsetsduring signaling or in disease manifestations. Correlation of changes insignaling cascades can also find use in classifying pathologicdifferences in populations. The present invention handles these issuesby providing methods and compositions for simultaneously detecting theactivation state of a plurality of activatable elements (e.g. proteins)in single cells using flow cytometry and by providing analytical meansfor correlating such states to differences in populations.

The invention further provides methods and compositions of screening forbioactive agents capable of modulating the activation state ofactivatable elements in single cells. Thus the methods and compositionsare not only useful for predicting or diagnosing a disease state, butalso for monitoring treatment of a disease state. For example,determining the phosphorylation states of a set of proteins can allowfor the classification and clustering of subpopulations of cells intorelevant categories, which in turn aid in making determinations such asprognosis, disease progression, response to particular drugs, etc.

The invention also provides methods and compositions that include theuse of potentiators to amplify the amount of functional data that can begathered in connection with the activation state of particular elements.For example, the methods and compositions of the instant invention canbe used to determine the activation profile of cells derived from asingle source but which have been individually exposed to differentpotentiators (e.g. various cytokines), and changes to activationprofiles arising due to the influence of distinct potentiators can beincorporated into the analysis.

Activation

As used herein, an “activatable element” or grammatical equivalentsthereof, refers to a cellular element that has at least two isoform (andin some cases three or more isoforms) that corresponds to specific formsof the element having a particular biological, biochemical, or physicalproperties, e.g., an enzymatic activity, a modification (e.g.,post-translational modification), or a conformation. In preferredembodiments, the activatable element is a protein. While in general thediscussion below refers to activatable proteins, other activatablecellular elements are also included such as lipids, carbohydrates, andmetabolites as discussed further below. The activatable element can beactivated or nonactivated with respect to a particular biologicalactivity, modification, or conformation. Specifically, the “activated”or “active” isoform of the activatable protein has the particularbiological activity, modification, or conformation, whereas the“non-activated” or “non-active” isoform of the activatable protein doesnot have (or has a lesser or diminished level of) the particularbiological activity, modification, or conformation, respectively. Insome embodiments, there may be more than one isoform associated withactivity or activation state; for example, in the case of activatableenzymes there may be an isoform associated with an “open” conformationavailable for substrate binding, a second “transition state” isoform,and an isoform devoid of activity (e.g., where the activity isinhibited). Similarly, certain proteins may have multiplephosphorylation states, multiple glycosylation states, etc. Thus theactivatable elements generally have an “inactive” isoform and at leastone “active” isoform. In some cases, however, it may be desirable toinclude elements in the analysis that do no have isoforms; for example,one readout could be presence or absence of a particular metabolite,e.g. there may not be an “activatable” state.

In a preferred embodiment, the biological, biochemical, or physicalproperty (e.g. enzymatic activity, modification, or conformation) of theactivatable element is inducible or “activatable” by an activating agentor by cell signaling events. Examples of activating agents include, butare not limited to, kinases, phosphatases, proteases (e.g., caspases),drugs and hormones. Examples of cell signaling events include, but arenot limited to, element clustering or binding of a cognate molecule orligand. These examples, among others, are discussed in detail below.

The instant invention also makes use of cells that have been“potentiated.” In contrast to “activation,” a “potentiated” state refersthe state of a, cell after exposure to a potentiator which then can beactivated as the case may be. As described in detail below, potentiatorsexert their effect on signalling cascades by directly or indirectlyimpacting the ability of an activatable protein to switch betweenactivation isoforms. Examples of potentiators generally include a widevariety of environmental cues related to cellular activation, bothchemica/biochemical in nature (e.g. IIs and IFNs) or physical (thermal,pH, limited media, UV radiation, etc).

As used herein, an “isoform” or grammatical equivalents thereof, refersto a form of an activatable element having a specific detectablebiological activity, modification, or conformation. The isoform can bean activated (or active) form, or non-activated (or not active) form ofan activatable protein. Particular or specific properties or activitiesare generally associated with activated isoforms of activatable proteinsand will sometimes be referred to herein as “activation stateactivities.”

Types of Activation

Activation of an activatable protein can take a variety of differentforms and generally involves an alteration of the biological,biochemical, and/or physical properties of the protein. For example,many activatable proteins are dynamically regulated in response tocovalent modification of the protein. Other activation events may benon-covalent; for example, activation of many enzymes is subject toallosteric inhibition, which generally involves the non-covalent bindingof an inhibitor molecule.

As will be appreciated by those in the art, a wide variety of activationevents can find use in the present invention. In general, the basicrequirement is that the activation results in a change in theactivatable protein that is detectable by some indication (termed an“activation state indicator”), preferably by altered binding to alabeled binding element or by changes in detectable biologicalactivities (e.g., the activated state has an enzymatic activity whichcan be measured and compared to a lack of activity in the non-activatedstate). What is important is to differentiate, using detectable eventsor moieties, between the isoforms of two or more activation states (e.g.“off” and “on”).

Accordingly, the present invention provides for the detection of a widevariety of activation events, including, but not limited to,phosphorylation, cleavage, prenylation, intermolecular clustering,conformational changes, glycosylation, acetylation, cysteinylation,nitrosylation, methylation, ubiquination, sulfation, as well as theproduction of activated isoforms of selenoproteins and fusion proteins.

One example of covalent modification is the substitution of a phosphategroup for a hydroxyl group in the side chain of an amino acid(phosphorylation). A wide variety of proteins are known that recognizespecific protein substrates and catalyze the phosphorylation of serine,threonine, or tyrosine residues on their protein substrates. Suchproteins are generally termed “kinases.” Substrate proteins that arecapable of being phosphorylated are often referred to asphosphoproteins. Once phosphorylated, a substrate protein may have itsphosphorylated residue converted back to a hydroxyl one by the action ofa protein phosphatase that specifically recognizes the substrateprotein. Protein phosphatases catalyze the replacement of phosphategroups by hydroxyl groups on serine, threonine, or tyrosine residues.Through the action of kinases and phosphatases a protein may bereversibly or irreversibly phosphorylated on a multiplicity of residuesand its activity may be regulated thereby. Thus, the presence or absenceor absence of one or more phosphate groups in an activatable protein isa preferred readout in the present invention.

Another example of a covalent modification of an activatable protein isthe acetylation of histones. Through the activity of various acetylasesand deacetylylases the DNA binding function of histone proteins istightly regulated. Furthermore, histone acetylation and histonedeactelyation have been linked with malignant progression. See Nature,2004 May 27; 429(6990): 457-63.

Another form of activation involves cleavage of the activatable element.For example, one form of protein regulation involves proteolyticcleavage of a peptide bond. While random or misdirected proteolyticcleavage may be detrimental to the activity of a protein, many proteinsare activated by the action of proteases that recognize and cleavespecific peptide bonds. Many proteins derive from precursor proteins, orpro-proteins, which give rise to a mature isoform of the proteinfollowing proteolytic cleavage of specific peptide bonds. Many growthfactors are synthesized and processed in this manner, with a matureisoform of the protein typically possessing a biological activity notexhibited by the precursor form. Many enzymes are also synthesized andprocessed in this manner, with a mature isoform of the protein typicallybeing enzymatically active, and the precursor form of the protein beingenzymatically inactive. This type of regulation is generally notreversible. Accordingly, to inhibit the activity of a proteolyticallyactivated protein, mechanisms other than “reattachment” must be used.For example, many proteolytically activated proteins are relativelyshort-lived proteins, and their turnover effectively results indeactivation of the signal. Inhibitors may also be used. Among theenzymes that are proteolytically activated are serine and cysteineproteases, including cathepsins and caspases.

In a preferred embodiment, the activatable enzyme is a caspase. Thecaspases are an important class of proteases that mediate programmedcell death (referred to in the art as “apoptosis”). Caspases areconstitutively present in most cells, residing in the cytosol as asingle chain proenzyme. These are activated to fully functionalproteases by a first proteolytic cleavage to divide the chain into largeand small caspase subunits and a second cleavage to remove theN-terminal domain. The subunits assemble into a tetramer with two activesites (Green, Cell 94:695-698, 1998). Many other proteolyticallyactivated enzymes, known in the art as “zymogens,” also find use in theinstant invention as activatable elements.

In an alternative embodiment the activation of the activatable elementinvolves prenylation of the element. By “prenylation”, and grammaticalequivalents used herein, is meant the addition of any lipid group to theelement. Common examples of prenylation include the addition of farnesylgroups, geranylgeranyl groups, myristoylation and palmitoylation. Ingeneral these groups are attached via thioether linkages to theactivatable element, although other attachments may be used. Inalternative embodiment, activation of the activatable element isdetected as intermolecular clustering of the activatable element. By“clustering” or “multimerization”, and grammatical equivalents usedherein, is meant any reversible or irreversible association of one ormore signal transduction elements. Clusters can be made up of 2, 3, 4,etc., elements. Clusters of two elements are termed dimers. Clusters of3 or more elements are generally termed oligomers, with individualnumbers of clusters having their own designation; for example, a clusterof 3 elements is a trimer, a cluster of 4 elements is a tetramer, etc.

Clusters can be made up of identical elements or different elements.Clusters of identical elements are termed “homo” clusters, whileclusters of different elements are termed “hetero” clusters Accordingly,a cluster can be a homodimer, as is the case for the β₂-adrenergicreceptor.

Alternatively, a cluster can be a heterodimer, as is the case forGABA_(B)-R. In other embodiments, the cluster is a homotrimer, as in thecase of TNFα, or a heterotrimer such the one formed by membrane-boundand soluble CD95 to modulate apoptosis. In further embodiments thecluster is a homo-oligomer, as in the case of Thyrotropin releasinghormone receptor, or a hetero-oligomer, as in the case of TGFβ1.

In a preferred embodiment, the activation or signaling potential ofelements is mediated by clustering, irrespective of the actual mechanismby which the element's clustering is induced. For example, elements canbe activated to cluster a) as membrane bound receptors by binding toligands (ligands including both naturally occurring or syntheticligands), b) as membrane bound receptors by binding to other surfacemolecules, or c) as intracellular (non-membrane bound) receptors bindingto ligands.

In a preferred embodiment the activatable elements are membrane boundreceptor elements that cluster upon ligand binding such as cell surfacereceptors. As used herein, “cell surface receptor” refers to moleculesthat occur on the surface of cells, interact with the extracellularenvironment, and transmit or transduce the information regarding theenvironment intracellularly in a manner that may modulate cellularactivity directly or indirectly, e.g., via intracellular secondmessenger activities or transcription of specific promoters, resultingin transcription of specific genes. One class of receptor elementsincludes membrane bound proteins, or complexes of proteins, which areactivated to cluster upon ligand binding. As is known in the art, thesereceptor elements can have a variety of forms, but in general theycomprise at least three domains. First, these receptors have aligand-binding domain, which can be oriented either extracellularly orintracellularly, usually the former. Second, these receptors have amembrane-binding domain (usually a transmembrane domain), which can takethe form of a seven pass transmembrane domain (discussed below inconnection with G-protein-coupled receptors) or a lipid modification,such as myristylation, to one of the receptor's amino acids which allowsfor membrane association when the lipid inserts itself into the lipidbilayer. Finally, the receptor has an signaling domain, which isresponsible for propagating the downstream effects of the receptor.Examples of such receptor elements include hormone receptors, cytokinereceptors, steroid receptors, adhesion receptors and growth factorreceptors, including, but not limited to, PDGF-R (platelet derivedgrowth factor receptor), EGF-R (epidermal growth factor receptor),VEGF-R (vascular endothelial growth factor), uPAR (urokinase plasminogenactivator receptor), ACHR (acetylcholine receptor), IgE-R(immunoglobulin E receptor), estrogen receptor, thyroid hormonereceptor, integrin receptors (β1, β2, β3, β4, β5, β6, α1, α2, α3, α4,α5, α6), MAC-1 (β2 and cd11b), αVβ3, opiod receptors (mu and kappa), FCreceptors, serotonin receptors (5-HT, 5-HT6, 5-HT7), β-adrenergicreceptors, insulin receptor, leptin receptor, TNF receptor(tissue-necrosis factor), cytokine receptors (IL1-a, IL-b, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10. IL-12, IL-15, IL-18, IL-21,CCR5, CCR7, CXCR4, CCR-1-10, CCL20), statin receptors, FAS receptor,BAFF receptor, FLT3 receptor, GMCSF receptor, and fibronectin receptor.

In a preferred embodiment the activatable element is a cytokinereceptor. Cytokines are a family of soluble mediators of cell-to-cellcommunication that includes interleukins, interferons, andcolony-stimulating factors. The characteristic features of cytokines liein their functional redundancy and pleiotropy. Most of the cytokinereceptors that constitute distinct superfamilies do not possessintrinsic protein tyrosine kinase domains, yet receptor stimulationusually invokes rapid tyrosine phosphorylation of intracellularproteins, including the receptors themselves. Many members of thecytokine receptor superfamily activate the Jak protein tyrosine kinasefamily, with resultant phosphorylation of the STAT transcriptionalactivator factors. IL-2, IL-4, IL-7 and Interferon γ have all been shownto activate Jak kinases (Frank et al. (1995) Proc. Natl. Acad. Sci. USA92:7779-7783); Scharfe et al. (1995) Blood 86:2077-2085); (Bacon et al.(1995) Proc. Natl. Acad. Sci. USA 92:7307-7311); and (Sakatsume et al.(1995) J. Biol. Chem. 270:17528-17534). Events downstream of Jakphosphorylation have also been elucidated. For example, exposure of Tlymphocytes to IL-2 has been shown to lead to the phosphorylation ofsignal transducers and activators of transcription (STAT) proteinsSTAT1α, STAT1β, and STAT3, as well as of two STAT-related proteins, p94and p95. The STAT proteins were found to translocate to the nucleus andto bind to a specific DNA sequence, thus suggesting a mechanism by whichIL-2 may activate specific genes involved in immune cell function (Franket al. supra). Jak3 is associated with the gamma chain of the IL-2,IL-4, and IL-7 cytokine receptors (Fujii et al., (1995) Proc. Natl.Acad. Sci. 92:5482-5486) and (Musso et al. (1995) J. Exp. Med.181:1425-1431). The Jak kinases have been shown to be activated bynumerous ligands that signal via cytokine receptors such as, growthhormone, erythropoietin and IL-6 (Kishimoto (1994) Stem cells Suppl.12:37-44).

In a preferred embodiment the activatable element is a member of thenerve growth factor receptor superfamily, such as the Tumor necrosisfactor α receptor. Tumor necrosis factor α (TNF-α or TNF-alpha) is apleiotropic cytokine that is primarily produced by activated macrophagesand lymphocytes but is also expressed in endothelial cells and othercell types. TNF-alpha is a major mediator of inflammatory,immunological, and pathophysiological reactions. (Grell, M., et al.,(1995) Cell, 83:793-802). Two distinct forms of TNF exist, a 26 kDamembrane expressed form and the soluble 17 kDa cytokine which is derivedfrom proteolytic cleavage of the 26 kDa form. The soluble TNFpolypeptide is 157 amino acids long and is the primary biologicallyactive molecule.

TNF-alpha exerts its biological effects through interaction withhigh-affinity cell surface receptors. Two distinct membrane TNF-alphareceptors have been cloned and characterized. These are a 55 kDaspecies, designated p55 TNF-R and a 75 kDa species designated p75 TNF-R(Corcoran. A. E., et al., (1994) Eur. J. Biochem., 223:831-840). The twoTNF receptors exhibit 28% similarity at the amino acid level. This isconfined to the extracellular domain and consists of four repeatingcysteine-rich motifs, each of approximately 40 amino acids. Each motifcontains four to six cysteines in conserved positions. Dayhoff analysisshows the greatest intersubunit similarity among the first three repeatsin each receptor. This characteristic structure is shared with a numberof other receptors and cell surface molecules, which comprise theTNF-R/nerve growth factor receptor superfamily (Corcoran. A. E., et al.,(1994) Eur. J. Biochem., 223:831-840).

TNF signaling is initiated by receptor clustering, either by thetrivalent ligand TNF or by cross-linking monoclonal antibodies(Vandevoorde, V., et al., (1997) J. Cell Biol., 137:1627-1638).Crystallographic studies of TNF and the structurally related cytokine,lymphotoxin (LT), have shown that both cytokines exist as homotrimers,with subunits packed edge to edge in threefold symmetry. Structurally,neither TNF or LT reflect the repeating pattern of the their receptors.Each monomer is cone shaped and contains two hydrophilic loops onopposite sides of the base of the cone. Recent crystal structuredetermination of a p55 soluble TNF-R/LT complex has confirmed thehypothesis that loops from adjacent monomers join together to form agroove between monomers and that TNF-R binds in these grooves (Corcoran.A. E., et al., (1994) Eur. J. Biochem., 223:831-840).

In preferred embodiment, the activatable element is a receptor tyrosinekinase. The receptor tyrosine kinases can be divided into five subgroupson the basis of structural similarities in their extracellular domainsand the organization of the tyrosine kinase catalytic region in theircytoplasmic domains. Sub-groups I (epidermal growth factor (EGF)receptor-like), II (insulin receptor-like) and the EPH/ECK familycontain cysteine-rich sequences (Hirai et al., (1987) Science238:1717-1720 and Lindberg and Hunter, (1990) Mol. Cell. Biol.10:6316-6324). The functional domains of the kinase region of thesethree classes of receptor tyrosine kinases are encoded as a contiguoussequence (Hanks et al. (1988) Science 241:42-52). Subgroups III(platelet-derived growth factor (PDGF) receptor-like) and IV (thefibro-blast growth factor (FGF) receptors) are characterized as havingimmunoglobulin (Ig)-like folds in their extracellular domains, as wellas having their kinase domains divided in two parts by a variablestretch of unrelated amino acids (Yanden and Ullrich (1988) supra andHanks et al. (1988) supra).

The family with by far the largest number of known members is the Ephfamily (with the first member of the family originally isolated from anerythropoietin producing hepatocellular carcinoma cell line). Since thedescription of the prototype, the Eph receptor (Hirai et al. (1987)Science 238:1717-1720), sequences have been reported for at least tenmembers of this family, not counting apparently orthologous receptorsfound in more than one species. Additional partial sequences, and therate at which new members are still being reported, suggest the familyis even larger (Maisonpierre et al. (1993) Oncogene 8:3277-3288; Andreset al. (1994) Oncogene 9:1461-1467; Henkemeyer et al. (1994) Oncogene9:1001-1014; Ruiz et al. (1994) Mech. Dev. 46:87-100; Xu et al. (1994)Development 120:287-299; Zhou et al. (1994) J. Neurosci. Res.37:129-143; and references in Tuzi and Gullick (1994) Br. J. Cancer69:417-421). Remarkably, despite the large number of members in the Ephfamily, all of these molecules were identified as orphan receptorswithout known ligands.

As used herein, the terms “Eph receptor” or “Eph-type receptor” refer toa class of receptor tyrosine kinases, comprising at least elevenparalogous genes, though many more orthologs exist within this class,e.g. homologs from different species. Eph receptors, in general, are adiscrete group of receptors related by homology, and easilyrecognizable, e.g., they are typically characterized by an extracellulardomain containing a characteristic spacing of cysteine residues near theN-terminus and two fibronectin type III repeats (Hirai et al. (1987)Science 238:1717-1720; Lindberg et al. (1990) Mol. Cell. Biol.10:6316-6324; Chan et al. (1991) Oncogene 6:1057-1061; Maisonpierre etal. (1993) Oncogene 8:3277-3288; Andres et al. (1994) Oncogene9:1461-1467; Henkemeyer et al. (1994) Oncogene 9:1001-1014; Ruiz et al.(1994) Mech. Dev. 46:87-100; Xu et al. (1994) Development 120:287-299;Zhou et al. (1994) J. Neurosci. Res. 37:129-143; and references in Tuziand Gullick (1994) Br. J. Cancer 69:417-421). Exemplary Eph receptorsinclude the eph, elk, eck, sek, mek4, hek, hek2, eek, erk, tyro1, tyro4,tyro5, tyro6, tyro11, cek4, cek5, cek6, cek7, cek8, cek9, cek10, bsk,rtk1, rtk2, rtk3, myk1, myk2, ehk1, ehk2, pagliaccio, htk, erk and nukreceptors.

In another embodiment the receptor element is a member of thehematopoietin receptor superfamily. Hematopoietin receptor superfamilyis used herein to define single-pass transmembrane receptors, with athree-domain architecture: an extracellular domain that binds theactivating ligand, a short transmembrane segment, and a domain residingin the cytoplasm. The extracellular domains of these receptors have lowbut significant homology within their extracellular ligand-bindingdomain comprising about 200-210 amino acids. The homologous region ischaracterized by four cysteine residues located in the N-terminal halfof the region, and a Trp-Ser-X-Trp-Ser (WSXWS) motif located justoutside the membrane-spanning domain. Further structural and functionaldetails of these receptors are provided by Cosman, D. et al., (1990).The receptors of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, prolactin,placental lactogen, growth hormone GM-CSF, G-CSF, M-CSF anderythropoietin have, for example, been identified as members of thisreceptor family.

In a further embodiment, the receptor element is an integrin other thanLeukocyte Function Antigen-1 (LFA-1). Members of the integrin family ofreceptors function as heterodimers, composed of various α and βsubunits, and mediate interactions between a cell's cytoskeleton and theextracellular matrix. (Reviewed in, Giancotti and Ruoslahti, Science285, 13 Aug. 1999). Different combinations of the α and β subunits giverise to a wide range of ligand specificities, which may be increasedfurther by the presence of cell-type-specific factors. Integrinclustering is know to activate a number of intracellular signals, suchas RAS, MAP kinase, and phosphotidylinosital-3-kinase. In a preferredembodiment the receptor element is a heterodimer (other than LFA-1)composed of a β integrin and an α integrin chosen from the followingintegrins; β1, β2, β3, β4, β35, β36, α1, α2, α3, α4, α5, and α6, or isMAC-1 (β2 and cd11b), or αVβ₃.

In another embodiment the activatable elements cluster for signaling bycontact with other surface molecules. In contrast to the receptorsdiscussed above, these elements cluster for signaling by contact withother surface molecules, and generally use molecules presented on thesurface of a second cell as ligands. Receptors of this class areimportant in cell-cell interactions, such mediating cell-to-celladhesion and immunorecognition.

Examples of such receptor elements are CD3 (T cell receptor complex),BCR (B cell receptor complex), CD4, CD28, CD80, CD86, CD54, CD102, CD50and ICAMs 1, 2 and 3.

In a preferred embodiment the receptor element is a T cell receptorcomplex (TCR). TCRs occur as either of two distinct heterodimers, αβ orγδ, both of which are expressed with the non-polymorphic CD3polypeptides γ, δ, ε, ζ. The CD3 polypeptides, especially ζ and itsvariants, are critical for intracellular signaling. The αβ TCRheterodimer expressing cells predominate in most lymphoid compartmentsand are responsible for the classical helper or cytotoxic T cellresponses. Im most cases, the αβ TCR ligand is a peptide antigen boundto a class I or a class II MHC molecule (Fundamental Immunology, fourthedition, W. E. Paul, ed., Lippincott-Raven Publishers, 1999, Chapter 10,pp 341-367).

In a preferred embodiment the receptor element is a B cell antigenreceptor (BCR). Antigen contact with a specific B cell triggers thetransmembrane signaling function of the BCR. BCR molecules are rapidlyinternalized after antigen binding, leading to antigen uptake anddegradation in endosomes or lysosomes. In the case of protein antigens,antigen-derived peptides bind in the groove of class II MHC molecules.Upon binding, this complex is sent to the cell surface, where it servesas a stimulus for specific helper T cells. Antigen recognition by thehelper T cell induces it to form a tight and long lasting interactionwith the B cell and to synthesize B cell growth and differentiationfactors. B cells activated in this way may proliferate and terminallydifferentiate to antibody secreting cells (also called plasma cells)(Fundamental Immunology, fourth edition, W. E. Paul, ed.,Lippincott-Raven Publishers, 1999, Chapters 6-7, pp 183-261)

In a preferred embodiment the element is an intracellular adhesionmolecule (ICAM). ICAMs-1, -2, and -3 are cellular adhesion moleculesbelonging to the immunogloblin superfamily. Each of these receptors hasa single membrane-spanning domain and all bind to β2 integrins viaextracellular binding domains similar in structure to Ig-loops. (SignalTransduction, Gomperts, et al., eds, Academic Press Publishers, 2002,Chapter 14, pp 318-319).

In another embodiment, the activatable element is a member of the largefamily of G-protein-coupled receptors. It has recently been reportedthat a G-protein-coupled receptors are capable of clustering. (Kroeger,et al., J Biol Chem 276:16, 12736-12743, Apr. 20, 2001; Bai, et al., JBiol Chem 273:36, 23605-23610, Sep. 4, 1998; Rocheville, et al., J BiolChem 275 (11), 7862-7869, Mar. 17, 2000). As used hereinG-protein-coupled receptor, and grammatical equivalents thereof, refersto the family of receptors that bind to heterotrimeric “G proteins.”Many different G proteins are known to interact with receptors. Gprotein signaling systems include three components: the receptor itself,a GTP-binding protein (G protein), and an intracellular target protein.The cell membrane acts as a switchboard. Messages arriving throughdifferent receptors can produce a single effect if the receptors act onthe same type of G protein. On the other hand, signals activating asingle receptor can produce more than one effect if the receptor acts ondifferent kinds of G proteins, or if the G proteins can act on differenteffectors.

In their resting state, the G proteins, which consist of alpha (α), beta(β) and gamma (γ) subunits, are complexed with the nucleotide guanosinediphosphate (GDP) and are in contact with receptors. When a hormone orother first messenger binds to a receptor, the receptor changesconformation and this alters its interaction with the G protein. Thisspurs the α subunit to release GDP, and the more abundant nucleotideguanosine triphosphate (GTP), replaces it, activating the G protein. TheG protein then dissociates to separate the α subunit from the stillcomplexed beta and gamma subunits. Either the Gα subunit, or the Gβγcomplex, depending on the pathway, interacts with an effector. Theeffector (which is often an enzyme) in turn converts an inactiveprecursor molecule into an active “second messenger,” which may diffusethrough the cytoplasm, triggering a metabolic cascade. After a fewseconds, the Gα converts the GTP to GDP, thereby inactivating itself.The inactivated Gα may then reassociate with the Gβγ complex.

Hundreds, if not thousands, of receptors convey messages throughheterotrimeric G proteins, of which at least 17 distinct forms have beenisolated. Although the greatest variability has been seen in the αsubunit, several different β and γ structures have been reported. Thereare, additionally, many different G protein-dependent effectors.

Most G protein-coupled receptors are comprised of a single protein chainthat is threaded through the plasma membrane seven times. Such receptorsare often referred to as seven-transmembrane receptors (STRs). More thana hundred different STRs have been found, including many distinctreceptors that bind the same ligand, and there are likely many more STRsawaiting discovery.

In addition, STRs have been identified for which the natural ligands areunknown; these receptors are termed “orphan” G protein-coupledreceptors, as described above. Examples include receptors cloned byNeote et al. (1993) Cell 72, 415; Kouba et al. FEBS Lett. (1993)321,173; and Birkenbach et al (1993) J. Virol. 67, 2209.

Known ligands for G protein coupled receptors include: purines andnucleotides, such as adenosine, cAMP, ATP, UTP, ADP, melatonin and thelike; biogenic amines (and related natural ligands), such as5-hydroxytryptamine, acetylcholine, dopamine, adrenaline, histamine,noradrenaline, tyramine/octopamine and other related compounds; peptidessuch as adrenocorticotrophic hormone (acth), melanocyte stimulatinghormone (msh), melanocortins, neurotensin (nt), bombesin and relatedpeptides, endothelins, cholecystokinin, gastrin, neurokinin b (nk3),invertebrate tachykinin-like peptides, substance k (nk2), substance p(nk1), neuropeptide y (npy), thyrotropin releasing-factor (trf),bradykinin, angiotensin ii, beta-endorphin, c5a anaphalatoxin,calcitonin, chemokines (also called intercrines), corticotrophicreleasing factor (crf), dynorphin, endorphin, fmlp and other formylatedpeptides, follitropin (fsh), fungal mating pheromones, galanin, gastricinhibitory polypeptide receptor (gip), glucagon-like peptides (glps),glucagon, gonadotropin releasing hormone (gnrh), growth hormonereleasing hormone (ghrh), insect diuretic hormone, interleukin-8,leutropin (1 h/hcg), met-enkephalin, opioid peptides, oxytocin,parathyroid hormone (pth) and pthrp, pituitary adenylyl cyclaseactivating peptide (pacap), secretin, somatostatin, thrombin,thyrotropin (tsh), vasoactive intestinal peptide (vip), vasopressin,vasotocin; eicosanoids such as ip-prostacyclin, pg-prostaglandins,tx-thromboxanes; retinal based compounds such as vertebrate 11-cisretinal, invertebrate 11-cis retinal and other related compounds; lipidsand lipid-based compounds such as cannabinoids, anandamide,lysophosphatidic acid, platelet activating factor, leukotrienes and thelike; excitatory amino acids and ions such as calcium ions andglutamate.

Preferred G protein coupled receptors include, but are not limited to:α1-adrenergic receptor, α1B-adrenergic receptor, α2-adrenergic receptor,α2B-adrenergic receptor, β1-adrenergic receptor, β2-adrenergic receptor,β3-adrenergic receptor, m1 acetylcholine receptor (AChR), m2 AChR, m3AChR, m4 AChR, m5 AChR, D1 dopamine receptor, D2 dopamine receptor, D3dopamine receptor, D4 dopamine receptor, D5 dopamine receptor, A1adenosine receptor, A2a adenosine receptor, A2b adenosine receptor, A3adenosine receptor, 5-HT1a receptor, 5-HT1b receptor, 5HT1-likereceptor, 5-HT1d receptor, 5HT1d-like receptor, 5HT1d beta receptor,substance K (neurokinin A) receptor, fMLP receptor (FPR), fMLP-likereceptor (FPRL-1), angiotensin II type 1 receptor, endothelin ETAreceptor, endothelin ETB receptor, thrombin receptor, growthhormone-releasing hormone (GHRH) receptor, vasoactive intestinal peptidereceptor, oxytocin receptor, somatostatin SSTR1 and SSTR2, SSTR3,cannabinoid receptor, follicle stimulating hormone (FSH) receptor,leutropin (LH/HCG) receptor, thyroid stimulating hormone (TSH) receptor,thromboxane A2 receptor, platelet-activating factor (PAF) receptor, C5aanaphylatoxin receptor, CXCR1 (IL-8 receptor A), CXCR2 (IL-8 receptorB), Delta Opioid receptor, Kappa Opioid receptor, mip-1alpha/RANTESreceptor (CRR1), Rhodopsin, Red opsin, Green opsin, Blue opsin,metabotropic glutamate mGluR1-6, histamine H2 receptor, ATP receptor,neuropeptide Y receptor, amyloid protein precursor receptor,insulin-like growth factor II receptor, bradykinin receptor,gonadotropin-releasing hormone receptor, cholecystokinin receptor,melanocyte stimulating hormone receptor, antidiuretic hormone receptor,glucagon receptor, and adrenocorticotropic hormone II receptor. Inaddition, there are at least five receptors (CC and CXC receptors)involved in HIV viral attachment to cells. The two major co-receptorsfor HIV are CXCR4, (fusin receptor, LESTR, SDF1 receptor) and CCR5(m-trophic). More preferred receptors include the following humanreceptors: melatonin receptor 1a, galanin receptor 1, neurotensinreceptor, adenosine receptor 2a, somatostatin receptor 2 andcorticotropin releasing factor receptor 1. Melatonin receptor 1a isparticularly preferred. Other G protein coupled receptors (GPCRs) areknown in the art.

In a preferred embodiment the activatable elements are intracellularreceptors capable of clustering. Elements of this class are notmembrane-bound. Instead, they are free to diffuse through theintracellular matrix where they bind soluble ligands prior to clusteringand signal transduction. In contrast to the previously describedelements, many members of this class are capable of binding DNA afterclustering to directly effect changes in RNA transcription.

In a preferred embodiment the intracellular receptors capable ofclustering are perioxisome proliferator-activated receptors (PPAR).PPARs are soluble receptors responsive to lipophillic compounds, andinduce various genes involved in fatty acid metabolism. The three PPARsubtypes, PPAR α, β, and γ have been shown to bind to DNA after ligandbinding and heterodimerization with retinoid X receptor. (Summanasekera,et al., J Biol Chem, M211261200, Dec. 13, 2002.)

In another embodiment the activatable element is a nucleic acid.Activation and deactivation of nucleic acids can occur in numerous waysincluding, but not limited to, cleavage of an inactivating leadersequence as well as covalent or non-covalent modifications that inducestructural or functional changes. For example, many catalytic RNAs, e.g.hammerhead ribozymes, can be designed to have an inactivating leadersequence that deactivates the catalitic activity of the ribozyme untilcleavage occurs. An example of a covalent modification is methylation ofDNA. Deactivation by methylation has been shown to be a factor in thesilencing of certain genes, e.g. STAT regulating SOCS genes inlymphomas. See Leukemia See 2004 February; 18(2): 356-8. SOCS1 and SHP1hypermethylation in mantle cell lymphoma and follicular lymphoma:implications for epigenetic activation of the Jak/STAT pathway. Chim CS, Wong K Y, Loong F, Srivastava G.

In another embodiment the activatable element is a small molecule,carbohydrate, lipid or other naturally occurring or synthetic compoundcapable of having an activated isoform. In addition, as pointed outabove, activation of these elements need not include switching from oneform to another, but can be detected as the presence or absence of thecompound. For example, activation of cAMP (cyclic adenosinemono-phosphate) can be detected as the presence of cAMP rather than theconversion from non-cyclic AMP to cyclic AMP.

Potentiation

In one embodiment, the instant invention involves determining theactivation profile of cells after the introduction of an environmentalcue (e.g. by administering a potentiator). In such an embodiment it isnot only the absolute level of activation that is determined, but alsothe role signalling cascades play in the activation of elements. Ingeneral, potentiators are used to alter signaling cascades prior todetermination of the activation profile of a cell, and the impact of thepotentiator is measured as differences in the activation profile ascompared to a non-potentiated cell. For example, by simultaneouslymeasuring multiple phosphorylation events at the single cell levelacross a complex population of tumor cells, it is possible to build anddistinguish pathway anomalies in different cell subsets. Similarly,potentiation can be viewed as “priming” of cells. One example of primingis the development of naïve cells into memory cells. One consequence ofthis priming is that memory cells respond faster as their thresholds foractivation are lower than naïve cells, due to their previous exposure tothe priming input.

Key to understanding the role of potentiators in the instant inventionis to appreciate the fact that an activation profile of any one cell orpopulation of cells includes the determination of the activation stateof one or more different activatable elements. Each of these elementsforms a specific node in a signalling cascade or network. As manycellular signalling networks are interconnected, cues that impact onenode can have an effect on the ability of other nodes to switch betweenactivation states. Therefore, by investigating the activation state ofvarious nodes in the presence or absence of various potentiators it ispossible to determine the underlying network structures interconnectingthe nodes and to categorize activation profiles by their impact on suchunderlying structures. Potentiation and its role in categorizingactivation profiles is discussed in detail under the heading “Analysis”below and in Example 2.

As pointed out above, potentiators can take the form of a wide varietyof environmental cues and inputs. The defining characteristic of apotentiator is that is a cue or input capable of impacting cellularsignalling networks. Examples of potentiators include, but are notlimited to, physical parameters such as heat, cold, UV radiation, aswell as cytokines, drugs, hormones, antibodies, peptides, and proteinfragments, either alone or in the context of cells, cells themselves,viruses, and biological and non-biological complexes (e.g. beads,plates, viral envelopes, antigen presentation molecules such as majorhistocompatibility complex).

In a preferred embodiment, the potentiator is a cell or cell population.For example, a common pool of normal T cells, thought of as effectors,can be exposed to various tumor cell populations from differentpatients, and the ability of the tumors to generate different signalingprofiles in the effectors (and of the effectors to reciprocallystimulate potentiated signaling in the various tumor cells) can bemonitored. Similarly, normal T cells from patients with a disease can bemixed with various tumor cell populations (from the same patient) andthe signaling potential (activation events) within the normal T cellsand the tumor cells can be profiled. Such assays could take place in asingle tube and the relevant cell sub-populations (e.g. effectors andtumor cells) identified using antibodies that recognize cell surfaceepitopes). Furthermore, these assays could take place under a series ofpotentiating conditions (e.g. co-stimulation using antibodies).

Detection of State

In general, there are a variety of ways to detect the activation stateof a particular protein (i.e. activatable element). In one embodiment,labeled biniding elements (“BEs”) are used, which bind specifically toone isoform of the protein. Alternatively the state of the activatableprotein is used for the readout; for example, in the case of cellsurface receptors with signalling domains, the activity (or lackthereof) of the signalling domain can be assayed directly. For example,the two isoforms may be no activity (negative signal) versus kinaseactivity (measured using chromogenic substrates).

Binding Elements

By “binding element,” “BE,” and grammatical equivalents thereof, ismeant any molecule, e.g., nucleic acids, small organic molecules, andproteins which are capable of detecting one isoform of an element overanother.

In a preferred embodiment, the BE is a protein, as used herein, the term“protein” means at least two covalently attached amino acids, whichincludes proteins, polypeptides, oligopeptides and peptides. The proteinmay be made up of naturally occurring amino acids and peptide bonds, orsynthetic peptidomimetic structures. Thus “amino acid”, or “peptideresidue”, as used herein means both naturally occurring and syntheticamino acids. For example, homo-phenylalanine, citrulline and noreleucineare considered amino acids for the purposes of the invention. “Aminoacid” also includes imino acid residues such as proline andhydroxyproline. The side chains may be in either the (R) or the (S)configuration. In the preferred embodiment, the amino acids are in the(S) or L-configuration. If non-naturally occurring side chains are used,non-amino acid substituents may be used, for example to prevent orretard in vivo degradation. Proteins including non-naturally occurringamino acids may be synthesized or in some cases, made recombinantly; seevan Hest et al., FEBS Lett 428:(1-2) 68-70 May 22, 1998 and Tang et al.,Abstr. Pap Am. Chem. S218: U138 Part 2 Aug. 22, 1999, both of which areexpressly incorporated by reference herein.

In a preferred embodiment, the protein BE is an antibody. In aparticularly preferred embodiment, the protein BE is an activationstate-specific antibody. Accordingly, the methods and compositions ofthe present invention may be used to detect any particular elementisoform in a sample that is antigenically detectable and antigenicallydistinguishable from other isoforms of the activatable element that arepresent in the sample. For example, the activation state-specificantibodies of the present invention can be used in the present methodsto identify distinct signaling cascades of a subset or subpopulation ofcomplex cell populations; and the ordering of protein activation (e.g.,kinase activation) in potential signaling hierarchies.

By “antibody” herein is meant a protein consisting of one or morepolypeptides substantially encoded by all or part of the recognizedimmunoglobulin genes. The recognized immunoglobulin genes, for examplein humans, include the kappa (k), lambda (l), and heavy chain geneticloci, which together comprise the myriad variable region genes, and theconstant region genes mu (u), delta (d), gamma (g), sigma (e), and alpha(a) which encode the IgM, IgD, IgG, IgE, and IgA isotypes respectively.Antibody herein is meant to include full length antibodies and antibodyfragments, and may refer to a natural antibody from any organism, anengineered antibody, or an antibody generated recombinantly forexperimental, therapeutic, or other purposes as further defined below.The term “antibody” includes antibody fragments, as are known in theart, such as Fab, Fab′, F(ab′)2, Fv, scFv, or other antigen-bindingsubsequences of antibodies, either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAtechnologies. Particularly preferred are full length antibodies thatcomprise Fc variants as described herein. The term “antibody” comprisesmonoclonal and polyclonal antibodies. Antibodies can be antagonists,agonists, neutralizing, inhibitory, or stimulatory.

The antibodies of the present invention may be nonhuman, chimeric,humanized, or fully human. For a description of the concepts of chimericand humanized antibodies see Clark et al., 2000 and references citedtherein (Clark, 2000, Immunol Today 21:397-402). Chimeric antibodiescomprise the variable region of a nonhuman antibody, for example VH andVL domains of mouse or rat origin, operably linked to the constantregion of a human antibody (see for example U.S. Pat. No. 4,816,567). Ina preferred embodiment, the antibodies of the present invention arehumanized. By “humanized” antibody as used herein is meant an antibodycomprising a human framework region (FR) and one or more complementaritydetermining regions (CDR's) from a non-human (usually mouse or rat)antibody. The non-human antibody providing the CDR's is called the“donor” and the human immunoglobulin providing the framework is calledthe “acceptor”. Humanization relies principally on the grafting of donorCDRs onto acceptor (human) VL and VH frameworks (Winter U.S. Pat. No.5,225,539). This strategy is referred to as “CDR grafting”.“Backmutation” of selected acceptor framework residues to thecorresponding donor residues is often required to regain affinity thatis lost in the initial grafted construct (U.S. Pat. Nos. 5,530,101;5,585,089; 5,693,761; 5,693,762; 6,180,370; 5,859,205; 5,821,337;6,054,297; 6,407,213). The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region,typically that of a human immunoglobulin, and thus will typicallycomprise a human Fc region. Methods for humanizing non-human antibodiesare well known in the art, and can be essentially performed followingthe method of Winter and co-workers (Jones et al., 1986, Nature321:522-525; Riechmann et al., 1988, Nature 332:323-329; Verhoeyen etal., 1988, Science, 239:1534-1536). Additional examples of humanizedmurine monoclonal antibodies are also known in the art, for exampleantibodies binding human protein C(O'Connor et al., 1998, Protein Eng11:321-8), interleukin 2 receptor (Queen et al., 1989, Proc Natl AcadSci, USA 86:10029-33), and human epidermal growth factor receptor 2(Carter et al., 1992, Proc Natl Acad Sci USA 89:4285-9). In an alternateembodiment, the antibodies of the present invention may be fully human,that is the sequences of the antibodies are completely or substantiallyhuman. A number of methods are known in the art for generating fullyhuman antibodies, including the use of transgenic mice (Bruggemann etal., 1997, Curr Opin Biotechnol 8:455-458) or human antibody librariescoupled with selection methods (Griffiths et al., 1998, Curr OpinBiotechnol 9:102-108).

Specifically included within the definition of “antibody” areaglycosylated antibodies. By “aglycosylated antibody” as used herein ismeant an antibody that lacks carbohydrate attached at position 297 ofthe Fc region, wherein numbering is according to the EU system as inKabat. The aglycosylated antibody may be a deglycosylated antibody,which is an antibody for which the Fc carbohydrate has been removed, forexample chemically or enzymatically. Alternatively, the aglycosylatedantibody may be a nonglycosylated or unglycosylated antibody, that is anantibody that was expressed without Fc carbohydrate, for example bymutation of one or residues that encode the glycosylation pattern or byexpression in an organism that does not attach carbohydrates toproteins, for example bacteria.

Specifically included within the definition of “antibody” arefull-length antibodies that contain an Fc variant portion. By “fulllength antibody” herein is meant the structure that constitutes thenatural biological form of an antibody, including variable and constantregions. For example, in most mammals, including humans and mice, thefull length antibody of the IgG class is a tetramer and consists of twoidentical pairs of two immunoglobulin chains, each pair having one lightand one heavy chain, each light chain comprising immunoglobulin domainsVL and CL, and each heavy chain comprising immunoglobulin domains VH,Cg1, Cg2, and Cg3. In some mammals, for example in camels and llamas,IgG antibodies may consist of only two heavy chains, each heavy chaincomprising a variable domain attached to the Fc region. By “IgG” as usedherein is meant a polypeptide belonging to the class of antibodies thatare substantially encoded by a recognized immunoglobulin gamma gene. Inhumans this class comprises IgG1, IgG2, IgG3, and IgG4. In mice thisclass comprises IgG1, IgG2a, IgG2b, IgG3.

As used herein, the term “activation state-specific antibody” or“activation state antibody” or grammatical equivalents thereof, refer toan antibody that specifically binds to a corresponding and specificantigen. Preferably, the corresponding and specific antigen is aspecific isoform of an activatable element. The binding of theactivation state-specific antibody is also preferably indicative of aspecific activation state of a specific activatable element. Thus, inpreferred embodiments, the binding of an activation state-specificantibody to a corresponding isoform of an activatable element isindicative of the identity of that element as well as the activationstate of that element.

As pointed out above, activation state specific antibodies can be usedto detect kinase activity, however additional means for determiningkinase activation are provided by the present invention. For example,substrates that are specifically recognized by protein kinases andphosphorylated thereby are known. Antibodies that specifically bind tosuch phosphorylated substrates but do not bind to suchnon-phosphorylated substrates (phospho-substrate antibodies) may be usedto determine the presence of activated kinase in a sample.

In a further embodiment, an element activation profile is determinedusing a multiplicity of activation state antibodies that have beenimmobilized. Antibodies may be non-diffusibly bound to an insolublesupport having isolated sample-receiving areas (e.g. a microtiter plate,an array, etc.). The insoluble supports may be made of any compositionto which the compositions can be bound, is readily separated fromsoluble material, and is otherwise compatible with the overall method ofscreening. The surface of such supports may be solid or porous and ofany convenient shape. Examples of suitable insoluble supports includemicrotiter plates, arrays, membranes, and beads. These are typicallymade of glass, plastic (e.g., polystyrene), polysaccharides, nylon ornitrocellulose, Teflon™, etc. Microtiter plates and arrays areespecially convenient because a large number of assays can be carriedout simultaneously, using small amounts of reagents and samples. In somecases magnetic beads and the like are included.

The particular manner of binding of the composition is not crucial solong as it is compatible with the reagents and overall methods of theinvention, maintains the activity of the composition and isnondiffusable. Preferred methods of binding include the use ofantibodies (which do not sterically block either the ligand binding siteor activation sequence when the protein is bound to the support), directbinding to “sticky” or ionic supports, chemical crosslinking, thesynthesis of the antibody on the surface, etc. Following binding of theantibody, excess unbound material is removed by washing. The samplereceiving areas may then be blocked through incubation with bovine serumalbumin (BSA), casein or other innocuous protein or other moiety.

The antigenicity of an activated isoform of an activatable element isdistinguishable from the antigenicity of non-activated isoform of anactivatable element or from the antigenicity of an isoform of adifferent activation state. In a preferred embodiment, an activatedisoform of an element possesses an epitope that is absent in anon-activated isoform of an element, or vice versa. In another preferredembodiment, this difference is due to covalent addition of moieties toan element, such as phosphate moieties, or due to a structural change inan element, as through protein cleavage, or due to an otherwise inducedconformational change in an element which causes the element to presentthe same sequence in an antigenically distinguishable way. In anotherpreferred embodiment, such a conformational change causes an activatedisoform of an element to present at least one epitope that is notpresent in a non-activated isoform, or to not present at least oneepitope that is presented by a non-activated isoform of the element. Insome embodiments, the epitopes for the distinguishing antibodies arecentered around the active site of the element, although as is known inthe art, conformational changes in one area of an element may causealterations in different areas of the element as well.

Many antibodies, many of which are commercially available (for example,see Cell Signaling Technology's catalogue, the contents which areincorporated herein by reference) have been produced which specificallybind to the phosphorylated isoform of a protein but do not specificallybind to a non-phosphorylated isoform of a protein. Many such antibodieshave been produced for the study of signal transducing proteins that arereversibly phosphorylated. In particular, many such antibodies have beenproduced which specifically bind to phosphorylated, activated isoformsof protein kinases and are sometimes referred to herein as kinaseactivation state antibodies or grammatical equivalents thereof.Particularly preferred antibodies for use in the present inventioninclude: phospho-AKT Ser473 monoclonal anti-4E2, phospho-p44/42 MAPkinase (Thr202/Tyr204) monoclonal antibody, phospho-TYK2 (Tyr1054/1055)antibody, phospho-p38 MAP kinase (Thr180/Tyr182) monoclonal antibody28B10, phospho-PKC-PAN substrate antibody, phospho-PKA-substrate,phospho-SAPK/JNK (Thr183/Tyr185) G9 monoclonal antibody,phospho-tyrosine monoclonal antibody (P-tyr-100), p44/42 MAPK, p38 MAPK,JNK/SAPK, and phospho-AKT-Thr308.

In a preferred embodiment, an epitope-recognizing fragment of anactivation state antibody rather than the whole antibody is used. Inanother preferred embodiment, the epitope-recognizing fragment isimmobilized. In another preferred embodiment, the antibody light chainthat recognizes an epitope is used. A recombinant nucleic acid encodinga light chain gene product that recognizes an epitope may be used toproduce such an antibody fragment by recombinant means well known in theart.

Non-activation state antibodies may also be used in the presentinvention. In a preferred embodiment, non-activation state antibodiesbind to epitopes in both activated and non-activated forms of anelement. Such antibodies may be used to determine the amount ofnon-activated plus activated element in a sample. In another preferredembodiment, non-activation state antibodies bind to epitopes present innon-activated forms of an element but absent in activated forms of anelement. Such antibodies may be used to determine the amount ofnon-activated element in a sample. Both types of non-activation stateantibodies may be used to determine if a change in the amount ofactivation state element, for example from samples before and aftertreatment with a candidate bioactive agent as described herein, coincidewith changes in the amount of non-activation state element. For example,such antibodies can be used to determine whether an increase inactivated element is due to activation of non-activation state element,or due to increased expression of the element, or both.

In another preferred embodiment, antibodies are immobilized using beadsanalogous to those known and used for standardization in flow cytometry.Attachment of a multiplicity of activation state specific antibodies tobeads may be done by methods known in the art and/or described herein.Such conjugated beads may be contacted with sample, preferably cellextract, under conditions that allow for a multiplicity of activatedelements, if present, to bind to the multiplicity of immobilizedantibodies. A second multiplicity of antibodies comprisingnon-activation state antibodies which are uniquely labeled may be addedto the immobilized activation state specific antibody-activated elementcomplex and the beads may be sorted by FACS on the basis of the presenceof each label, wherein the presence of label indicates binding ofcorresponding second antibody and the presence of correspondingactivated element.

In alternative embodiments of the instant invention, aromatic aminoacids of protein BEs may be replaced with D- or L-naphylalanine, D- orL-phenylglycine, D- or L-2-thieneylalanine, D- or L-1-, 2-, 3- or4-pyreneylalanine, D- or L-3-thieneylalanine, D- orL-(2-pyridinyl)-alanine, D- or L-(3-pyridinyl)-alanine, D- orL-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine,D-(trifluoromethyl)-phenylglycine, D-(trifluoromethyl)-phenylalanine,D-p-fluorophenylalanine, D- or L-p-biphenylphenylalanine, D- orL-p-methoxybiphenylphenylalanine, D- or L-2-indole(alkyl)alanines, andD- or L-alkylalanines where alkyl may be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, and non-acidic amino acids of C₁-C₂₀.

Acidic amino acids can be substituted with non-carboxylate amino acidswhile maintaining a negative charge, and derivatives or analogs thereof,such as the non-limiting examples of (phosphono)alanine, glycine,leucine, isoleucine, threonine, or serine; or sulfated (e.g., —SO₃H)threonine, serine, or tyrosine.

Other substitutions may include nonnatural hydroxylated amino acids maymade by combining “alkyl” with any natural amino acid. The term “alkyl”as used herein refers to a branched or unbranched saturated hydrocarbongroup of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl,isoptopyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl,hexadecyl, eicosyl, tetracisyl and the like. Alkyl includes heteroalkyl,with atoms of nitrogen, oxygen and sulfur. Preferred alkyl groups hereincontain 1 to 12 carbon atoms. Basic amino acids may be substituted withalkyl groups at any position of the naturally occurring amino acidslysine, arginine, ornithine, citrulline, or (guanidino)-acetic acid, orother (guanidino)alkyl-acetic acids, where “alkyl” is define as above.Nitrile derivatives (e.g., containing the CN-moiety in place of COOH)may also be substituted for asparagine or glutamine, and methioninesulfoxide may be substituted for methionine. Methods of preparation ofsuch peptide derivatives are well known to one skilled in the art.

In addition, any amide linkage in any of the polypeptides may bereplaced by a ketomethylene moiety. Such derivatives are expected tohave the property of increased stability to degradation by enzymes, andtherefore possess advantages for the formulation of compounds which mayhave increased in vivo half lives, as administered by oral, intravenous,intramuscular, intraperitoneal, topical, rectal, intraocular, or otherroutes.

Additional amino acid modifications of amino acids of variantpolypeptides of to the present invention may include the following:Cysteinyl residues may be reacted with alpha-haloacetates (andcorresponding amines), such as 2-chloroacetic acid or chloroacetamide,to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinylresidues may also be derivatized by reaction with compounds such asbromotrifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.Histidyl residues may be derivatized by reaction with compounds such asdiethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain, and para-bromophenacyl bromide mayalso be used; e.g., where the reaction is preferably performed in 0.1Msodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues may be reacted with compounds suchas succinic or other carboxylic acid anhydrides. Derivatization withthese agents is expected to have the effect of reversing the charge ofthe lysinyl residues.

Other suitable reagents for derivatizing alpha-amino-containing residuesinclude compounds such as imidoesters, e.g., as methyl picolinimidate;pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; andtransaminase-catalyzed reaction with glyoxylate. Arginyl residues may bemodified by reaction with one or several conventional reagents, amongthem phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrinaccording to known method steps. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group. The specific modification of tyrosyl residues perse is well known, such as for introducing spectral labels into tyrosylresidues by reaction with aromatic diazonium compounds ortetranitromethane.

N-acetylimidizol and tetranitromethane may be used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Carboxyl sidegroups (aspartyl or glutamyl) may be selectively modified by reactionwith carbodiimides (R′—N—C—N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermoreaspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues may be deamidated under mildly acidic conditions. Either formof these residues falls within the scope of the present invention.

In a preferred embodiment, the activation state-specific BE is a peptidecomprising a recognition structure that binds to a target structure onan activatable element. A variety of recognition structures are wellknown in the art and can be made using methods known in the art,including by phage display libraries (see e.g., Gururaja et al. Chem.Biol. (2000) 7:515-27; Houimel et al., Eur. J. Immunol. (2001)31:3535-45; Cochran et al. J. Am. Chem. Soc. (2001) 123:625-32; Houimelet al., Int. J. Cancer (2001) 92:748-55, each incorporated herein byreference). Alternatively, combinatorial chemistry methods may be usedfor producing recognition structures such as polymers with affinity fora target structure on an activatable protein (see e.g., Barn et al., J.Comb. Chem. (2001) 3:534-41; Ju et al., Biotechnol. (1999) 64:232-9,each expressly incorporated herein by reference). In an additionalembodiment, the recognition structure is an anti-laminin single-chainantibody fragment (scFv) (see e.g., Sanz et al., Gene Therapy (2002)9:1049-53; Tse et al., J. Mol. Biol. (2002) 317:85-94, each expresslyincorporated herein by reference). In a preferred embodiment, theactivation state-specific BE comprises the following recognitionstructure: SKVILFE—random peptide loop—SKVILFE. BEs having suchrecognition structures can bind with high affinity to specific targetstructures. Further, fluorophores can be attached to such BEs for use inthe methods of the present invention.

In a preferred embodiment the BE is a nucleic acid. By “nucleic acid” or“oligonucleotide” or grammatical equivalents herein means at least twonucleotides covalently linked together. A nucleic acid of the presentinvention will generally contain phosphodiester bonds, although in somecases, as outlined below, nucleic acid analogs are included that mayhave alternate backbones, comprising, for example, phosphoramide(Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein;Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J.Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487(1986); Sawai et al., Chem. Lett. 805 (1984), Letsinger et al., J. Am.Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:14191986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphosphoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al.,Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993);Carlsson et al., Nature 380:207 (1996), all of which are incorporated byreference). Other analog nucleic acids include those with positivebackbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995);non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240,5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed.English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470(1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994);Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modificationsin Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker etal., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogsare described in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of additional moieties such as labels, or to increase thestability and half-life of such molecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made.Alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.Particularly preferred are peptide nucleic acids (PNA) which includespeptide nucleic acid analogs. These backbones are substantiallynon-ionic under neutral conditions, in contrast to the highly chargedphosphodiester backbone of naturally occurring nucleic acids.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. As used herein, the term“nucleoside” includes nucleotides and nucleoside and nucleotide analogs,and modified nucleosides such as amino modified nucleosides. Inaddition, “nucleoside” includes non-naturally occurring analogstructures. Thus for example the individual units of a peptide nucleicacid, each containing a base, are referred to herein as a nucleoside.

Nucleic acid BEs may be naturally occurring nucleic acids, randomnucleic acids, or “biased” random nucleic acids. For example, digests ofprokaryotic or eukaryotic genomes may be used as is outlined above forproteins. Where the ultimate expression product is a nucleic acid, atleast 10, preferably at least 12, more preferably at least 15, mostpreferably at least 21 nucleotide positions need to be randomized, withmore preferable if the randomization is less than perfect. Similarly, ifthe ultimate expression product is an protein, at least 5, preferably atleast 6, more preferably at least 7 amino acid positions need to berandomized, again, more are preferable if the randomization is less thanperfect.

In a preferred embodiment, the BE is a synthetic compound. Any number oftechniques are available for the random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides. See for example WO 94/24314, herebyexpressly incorporated by reference, which discusses methods forgenerating new compounds, including random chemistry methods as well asenzymatic methods.

Alternatively, a preferred embodiment utilizes natural compounds, asBEs, in the form of bacterial, fungal, plant and animal extracts thatare available or readily produced.

Additionally, natural or synthetically produced compounds are readilymodified through conventional chemical, physical and biochemical means.Known pharmacological agents may be subjected to directed or randomchemical modifications, including enzymatic modifications, to produceBEs that may be used in the instant invention.

In another preferred embodiment the BE is a small organic compound. BEscan be synthesized from a series of substrates that can be chemicallymodified. “Chemically modified” herein includes traditional chemicalreactions as well as enzymatic reactions. These substrates generallyinclude, but are not limited to, alkyl groups (including alkanes,alkenes, alkynes and heteroalkyl), aryl groups (including arenes andheteroaryl), alcohols, ethers, amines, aldehydes, ketones, acids,esters, amides, cyclic compounds, heterocyclic compounds (includingpurines, pyrimidines, benzodiazepins, beta-lactams, tetracylines,cephalosporins, and carbohydrates), steroids (including estrogens,androgens, cortisone, ecodysone, etc.), alkaloids (including ergots,vinca, curare, pyrollizdine, and mitomycines), organometallic compounds,hetero-atom bearing compounds, amino acids, and nucleosides. Chemical(including enzymatic) reactions may be done on the moieties to form newsubstrates or BEs that can then be used in the present invention.

In a preferred embodiment the BE is a carbohydrate. As used herein theterm carbohydrate is meant to include any compound with the generalformula (CH₂O)_(n). Examples of preferred carbohydrates are di-, tri-and oligosaccharides, as well polysaccharides such as glycogen,cellulose, and starches.

In a preferred embodiment the BE is a lipid. As used herein the termlipid herein is meant to include any water insoluble organic moleculethat is soluble in nonpolar organic solvents. Examples of preferredlipids are steroids, such as cholesterol, and phospholipids such assphingomeylin.

Labels

The methods and compositions of the instant invention provide BEscomprising a label or tag. By label is meant a molecule that can bedirectly (i.e., a primary label) or indirectly (i.e., a secondary label)detected; for example a label can be visualized and/or measured orotherwise identified so that its presence or absence can be known. Acompound can be directly or indirectly conjugated to a label whichprovides a detectable signal, e.g. radioisotopes, fluorescers, enzymes,antibodies, particles such as magnetic particles, chemiluminescers, orspecific binding molecules, etc. Specific binding molecules includepairs, such as biotin and streptavidin, digoxin and antidigoxin etc.Preferred labels include, but are not limited to, optical fluorescentand chromogenic dyes including labels, label enzymes and radioisotopes.

Using the example of two activation state specific antibodies, by“uniquely labeled” is meant that a first activation state antibodyrecognizing a first activated element comprises a first label, andsecond activation state antibody recognizing a second activated elementcomprises a second label, wherein the first and second labels aredetectable and distinguishable, making the first antibody and the secondantibody uniquely labeled.

In general, labels fall into four classes: a) isotopic labels, which maybe radioactive or heavy isotopes; b) magnetic, electrical, thermallabels; c) colored, optical labels including luminescent, phosphorousand fluorescent dyes or moieties; and d) binding partners. Labels canalso include enzymes (horseradish peroxidase, etc.) and magneticparticles. In a preferred embodiment, the detection label is a primarylabel. A primary label is one that can be directly detected, such as afluorophore.

Preferred labels include optical labels such as fluorescent dyes ormoieties. Fluorophores can be either “small molecule” fluors, orproteinaceous fluors (e.g. green fluorescent proteins and all variantsthereof).

By “fluorescent label” is meant any molecule that may be detected viaits inherent fluorescent properties. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, TexasRed, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705 andOregon green. Suitable optical dyes are described in the 1996 MolecularProbes Handbook by Richard P. Haugland, hereby expressly incorporated byreference. Suitable fluorescent labels also include, but are not limitedto, green fluorescent protein (GFP; Chalfie, et al., Science263(5148):802-805 (Feb. 11, 1994); and EGFP; Clontech—Genbank AccessionNumber U55762), blue fluorescent protein (BFP; 1. QuantumBiotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor,Montreal (Quebec) Canada H3H 1J9; 2. Stauber, R. H. Biotechniques24(3):462-471 (1998); 3. Heim, R. and Tsien, R. Y. Curr. Biol. 6:178-182(1996)), enhanced yellow fluorescent protein (EYFP; 1. ClontechLaboratories, Inc., 1020 East Meadow Circle, Palo Alto, Calif. 94303),luciferase (Ichiki, et al., J. Immunol. 150(12):5408-5417 (1993)),3-galactosidase (Nolan, et al., Proc Natl Acad Sci USA 85(8):2603-2607(April 1988)) and Renilla WO 92/15673; WO 95/07463; WO 98/14605; WO98/26277; WO 99/49019; U.S. Pat. Nos. 5,292,658; 5,418,155; 5,683,888;5,741,668; 5,777,079; 5,804,387; 5,874,304; 5,876,995; and 5,925,558).All of the above-cited references are expressly incorporated herein byreference.

Particularly preferred labels for use in the present invention include:Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488,Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633,Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow andR-phycoerythrin (PE) (Molecular Probes) (Eugene, Oreg.), FITC,Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7(Amersham Life Science, Pittsburgh, Pa.). Tandem conjugate protocols forCy5PE, Cy5.5PE, Cy7PE, Cy5.5APC, Cy7APC are known in the art.Quantitation of fluorescent probe conjugation may be assessed todetermine degree of labeling and protocols including dye spectralproperties are also well known in the art.

In another preferred embodiment, the fluorescent label is a GFP and,more preferably, a Renilla, Ptilosarcus, or Aequorea species of GFP.

In a preferred embodiment, a secondary detectable label is used. Asecondary label is one that is indirectly detected; for example, asecondary label can bind or react with a primary label for detection,can act on an additional product to generate a primary label (e.g.enzymes), etc. Secondary labels include, but are not limited to, one ofa binding partner pair; chemically modifiable moieties; nucleaseinhibitors, enzymes such as horseradish peroxidase, alkalinephosphatases, lucifierases, etc.

In a preferred embodiment, the secondary label is a binding partnerpair. For example, the label may be a hapten or antigen, which will bindits binding partner. For example, suitable binding partner pairsinclude, but are not limited to: antigens (such as proteins (includingpeptides) and small molecules) and antibodies (including fragmentsthereof (FAbs, etc.)); proteins and small molecules, includingbiotin/streptavidin; enzymes and substrates or inhibitors; otherprotein-protein interacting pairs; receptor-ligands; and carbohydratesand their binding partners. Nucleic acid—nucleic acid binding proteinspairs are also useful. Preferred binding partner pairs include, but arenot limited to, biotin (or imino-biotin) and streptavidin, digeoxininand Abs, and Prolinx™ reagents.

In a preferred embodiment, the binding partner pair comprises an antigenand an antibody that will specifically bind to the antigen. By“specifically bind” herein is meant that the partners bind withspecificity sufficient to differentiate between the pair and othercomponents or contaminants of the system. The binding should besufficient to remain bound under the conditions of the assay, includingwash steps to remove non-specific binding. In some embodiments, thedissociation constants of the pair will be less than about 10⁻⁴ to 10⁻⁹M⁻¹, with less than about 10⁻⁵ to 10⁻⁹ M⁻¹ being preferred and less thanabout 10⁻⁷ to 10⁻⁹ M⁻¹ being particularly preferred.

In a preferred embodiment, the secondary label is a chemicallymodifiable moiety. In this embodiment, labels comprising reactivefunctional groups are incorporated into the molecule to be labeled. Thefunctional group can then be subsequently labeled (e.g. either before orafter the assay) with a primary label. Suitable functional groupsinclude, but are not limited to, amino groups, carboxy groups, maleimidegroups, oxo groups and thiol groups, with amino groups and thiol groupsbeing particularly preferred. For example, primary labels containingamino groups can be attached to secondary labels comprising aminogroups, for example using linkers as are known in the art; for example,homo- or hetero-bifunctional linkers as are well known (see 1994 PierceChemical Company catalog, technical section on cross-linkers, pages155-200, incorporated herein by reference).

In preferred embodiments, multiple fluorescent labels are employed inthe methods and compositions of the present invention. In a preferredembodiment, each label is distinct and distinguishable from otherlabels.

As will be appreciated in the art antibody-label conjugation may beperformed using standard procedures or by usingprotein-protein/protein-dye crosslinking kits from Molecular Probes(Eugene, Oreg.).

In a preferred embodiment labeled antibodies are used for functionalanalysis of activatable proteins in cells. In performing such analysisseveral areas of the experiment are considered: (1) identification ofthe proper combination of antibody cocktails for the stains (2),identification of the sequential procedure for the staining using theantigens (i.e., the activatable protein) and antibody clones ofinterest, and (3) thorough evaluation of cell culture conditions' effecton cell stimulation. Antigen clone selection is of particular importancefor surface antigens of human cells, as different antibody clones yielddifferent result and do not stain similarly in different protocols.Selection of cell types and optimization of culture conditions is also acritical component in detecting differences. For example, some celllines have the ability to adapt to culture conditions and can yieldheterogeneous responses.

The usage of multicolor, multiparameter flow cytometry requires primaryconjugated antibodies at defined fluorophores to protein (“FTP”) ratios.It is generally not sufficient to give a range of FTP ratios, but ratherit is necessary to quantitate the final product thoroughly as FTP ratiosdiffering in 2 molecules can represent significant decreases inphospho-epitope staining. It is also important to note that eachfluorophore's optimal FTP is unique and can differ amongst antibodyclones to phospho-epitopes.

In a preferred embodiment, the optimal ratio for any protein fluorophore(i.e. PE, APC, PE-TANDEM CONJUGATES (PE-TR, PE-Cy5, PE-Cy5.5, PE-Cy7,PE-Alexa colors (PE-AX610, PE-AX647, PE-680, PE-AX700, PE-AX750),APC-TANDEM CONJUGATES APC-AX680, APC-AX700, APC-AX750, APC-Cy5.5,APC-Cy7), GFP, BFP, CFP, DSRED, and all the derivates of the algaeproteins including the phycobilliproteins is 1:1 (one ab to one proteindye).

In additional embodiments, the FTP ratio is 1-6 for internal stains; forAX488 the FTP is preferably 2-5 and more preferably 4; for AX546 the FTPratio is preferably 2-6 and more preferably 2; for AX594 the FTP ratiois preferably 2-4; for AX633 the FTP is preferably 1-3; for AX647 theFTP ratio is preferably 1-4 and more preferably 2. For AX405, AX430,AX555, AX568, AX680, AX700, AX750 the FTP ratio is preferably 2-5.

Alternatively, detection systems based on FRET, discussed in detailbelow, may be used. FRET finds use in the instant invention, forexample, in detecting activation states that involve clustering ormultimerization wherein the proximity of two FRET labels is altered dueto activation. In a preferred embodiment, at least two fluorescentlabels are used which are members of a fluorescence resonance energytransfer (FRET) pair.

FRET is phenomenon known in the art wherein excitation of onefluorescent dye is transferred to another without emission of a photon.A FRET pair consists of a donor fluorophore and an acceptor fluorophore.The fluorescence emission spectrum of the donor and the fluorescenceabsorption spectrum of the acceptor must overlap, and the two moleculesmust be in close proximity. The distance between donor and acceptor atwhich 50% of donors are deactivated (transfer energy to the acceptor) isdefined by the Förster radius (Ro), which is typically 10-100 Å. Changesin the fluorescence emission spectrum comprising FRET pairs can bedetected, indicating changes in the number of that are in closeproximity (i.e., within 100 Å of each other). This will typically resultfrom the binding or dissociation of two molecules, one of which islabeled with a FRET donor and the other of which is labeled with a FRETacceptor, wherein such binding brings the FRET pair in close proximity.Binding of such molecules will result in an increased fluorescenceemission of the acceptor and/or quenching of the fluorescence emissionof the donor.

FRET pairs (donor/acceptor) useful in the invention include, but are notlimited to, EDANS/fluorescein, IAEDANS/fluorescein,fluorescein/tetramethylrhodamine, fluorescein/LC Red 640, fluorescein/Cy5, fluorescein/Cy 5.5 and fluorescein/LC Red 705.

In another aspect of FRET, a fluorescent donor molecule and anonfluorescent acceptor molecule (“quencher”) may be employed. In thisapplication, fluorescent emission of the donor will increase whenquencher is displaced from close proximity to the donor and fluorescentemission will decrease when the quencher is brought into close proximityto the donor. Useful quenchers include, but are not limited to, TAMRA,DABCYL, QSY 7 and QSY 33. Useful fluorescent donor/quencher pairsinclude, but are not limited to EDANS/DABCYL, Texas Red/DABCYL,BODIPY/DABCYL, Lucifer yellow/DABCYL, coumarin/DABCYL andfluorescein/QSY 7 dye.

The skilled artisan will appreciate that FRET and fluorescence quenchingallow for monitoring of binding of labeled molecules over time,providing continuous information regarding the time course of bindingreactions.

Preferably, changes in the degree of FRET are determined as a functionof the change in the ratio of the amount of fluorescence from the donorand acceptor moieties, a process referred to as “ratioing.” Changes inthe absolute amount of substrate, excitation intensity, and turbidity orother background absorbances in the sample at the excitation wavelengthaffect the intensities of fluorescence from both the donor and acceptorapproximately in parallel. Therefore the ratio of the two emissionintensities is a more robust and preferred measure of cleavage thaneither intensity alone.

The ratio-metric fluorescent reporter system described herein hassignificant advantages over existing reporters for protein integrationanalysis, as it allows sensitive detection and isolation of bothexpressing and non-expressing single living cells. In a preferredembodiment, the assay system uses a non-toxic, non-polar fluorescentsubstrate that is easily loaded and then trapped intracellularly.Modification of the fluorescent substrate by a cognate protein yields afluorescent emission shift as substrate is converted to product. Becausethe reporter readout is ratiometric it is unique among reporter proteinassays in that it controls for variables such as the amount of substrateloaded into individual cells. The stable, easily detected, intracellularreadout eliminates the need for establishing clonal cell lines prior toexpression analysis. This system and other analogous flow sortingsystems can be used to isolate cells having a particular receptorelement clustering and/or activation profile from pools of millions ofviable cells.

The methods and composition of the present invention may also make useof label enzymes. By label enzyme is meant an enzyme that may be reactedin the presence of a label enzyme substrate that produces a detectableproduct. Suitable label enzymes for use in the present invention includebut are not limited to, horseradish peroxidase, alkaline phosphatase andglucose oxidase. Methods for the use of such substrates are well knownin the art. The presence of the label enzyme is generally revealedthrough the enzyme's catalysis of a reaction with a label enzymesubstrate, producing an identifiable product. Such products may beopaque, such as the reaction of horseradish peroxidase with tetramethylbenzedine, and may have a variety of colors. Other label enzymesubstrates, such as Luminol (available from Pierce Chemical Co.), havebeen developed that produce fluorescent reaction products. Methods foridentifying label enzymes with label enzyme substrates are well known inthe art and many commercial kits are available. Examples and methods forthe use of various label enzymes are described in Savage et al.,Previews 247:6-9 (1998), Young, J. Virol. Methods 24:227-236 (1989),which are each hereby incorporated by reference in their entirety.

By radioisotope is meant any radioactive molecule. Suitableradioisotopes for use in the invention include, but are not limited to¹⁴C, ³H, ³²P, ³³P, ³⁵S, ¹²⁵I, and ¹³¹I. The use of radioisotopes aslabels is well known in the art.

As mentioned, labels may be indirectly detected, that is, the tag is apartner of a binding pair. By “partner of a binding pair” is meant oneof a first and a second moiety, wherein the first and the second moietyhave a specific binding affinity for each other. Suitable binding pairsfor use in the invention include, but are not limited to,antigens/antibodies (for example, digoxigenin/anti-digoxigenin,dinitrophenyl (DNP)/anti-DNP, dansyl-X-anti-dansyl,Fluorescein/anti-fluorescein, lucifer yellow/anti-lucifer yellow, andrhodamine anti-rhodamine), biotin/avidin (or biotin/streptavidin) andcalmodulin binding protein (CBP)/calmodulin. Other suitable bindingpairs include polypeptides such as the FLAG-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255: 192-194 (1992)]; tubulin epitope peptide [Skinner etal., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 proteinpeptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)] and the antibodies each thereto. As will beappreciated by those in the art, binding pair partners may be used inapplications other than for labeling, as is described herein.

As will be appreciated by those in the art, a partner of one bindingpair may also be a partner of another binding pair. For example, anantigen (first moiety) may bind to a first antibody (second moiety) thatmay, in turn, be an antigen for a second antibody (third moiety). Itwill be further appreciated that such a circumstance allows indirectbinding of a first moiety and a third moiety via an intermediary secondmoiety that is a binding pair partner to each.

As will be appreciated by those in the art, a partner of a binding pairmay comprise a label, as described above. It will further be appreciatedthat this allows for a tag to be indirectly labeled upon the binding ofa binding partner comprising a label. Attaching a label to a tag that isa partner of a binding pair, as just described, is referred to herein as“indirect labeling”.

By “surface substrate binding molecule” or “attachment tag” andgrammatical equivalents thereof is meant a molecule have bindingaffinity for a specific surface substrate, which substrate is generallya member of a binding pair applied, incorporated or otherwise attachedto a surface. Suitable surface substrate binding molecules and theirsurface substrates include, but are not limited to poly-histidine(poly-his) or poly-histidine-glycine (poly-his-gly) tags and Nickelsubstrate; the Glutathione-S Transferase tag and its antibody substrate(available from Pierce Chemical); the flu HA tag polypeptide and itsantibody 12CA5 substrate [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodysubstrates thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody substrate [Paborsky et al., Protein Engineering,3(6):547-553 (1990)]. In general, surface binding substrate moleculesuseful in the present invention include, but are not limited to,polyhistidine structures (His-tags) that bind nickel substrates,antigens that bind to surface substrates comprising antibody, haptensthat bind to avidin substrate (e.g., biotin) and CBP that binds tosurface substrate comprising calmodulin.

Production of antibody-embedded substrates is well known; see Slinkin etal., Bioconj. Chem., 2:342-348 (1991); Torchilin et al., supra;Trubetskoy et al., Bioconj. Chem. 3:323-327 (1992); King et al., CancerRes. 54:6176-6185 (1994); and Wilbur et al., Bioconjugate Chem.5:220-235 (1994) (all of which are hereby expressly incorporated byreference), and attachment of or production of proteins with antigens isdescribed above. Calmodulin-embedded substrates are commerciallyavailable, and production of proteins with CBP is described in Simcox etal., Strategies 8:40-43 (1995), which is hereby incorporated byreference in its entirety.

As will be appreciated by those in the art, tag-components of theinvention can be made in various ways, depending largely upon the formof the tag. Components of the invention and tags are preferably attachedby a covalent bond.

The production of tag-polypeptides by recombinant means when the tag isalso a polypeptide is described below. Production of tag-labeledproteins is well known in the art and kits for such production arecommercially available (for example, from Kodak and Sigma). Examples oftag labeled proteins include, but are not limited to, a Flag-polypeptideand His-polypeptide. Methods for the production and use of tag-labeledproteins are found, for example, in Winston et al., Genes and Devel.13:270-283 (1999), incorporated herein in its entirety, as well asproduct handbooks provided with the above-mentioned kits.

Biotinylation of target molecules and substrates is well known, forexample, a large number of biotinylation agents are known, includingamine-reactive and thiol-reactive agents, for the biotinylation ofproteins, nucleic acids, carbohydrates, carboxylic acids; see chapter 4,Molecular Probes Catalog, Haugland, 6th Ed. 1996, hereby incorporated byreference. A biotinylated substrate can be attached to a biotinylatedcomponent via avidin or streptavidin. Similarly, a large number ofhaptenylation reagents are also known (Id.).

Methods for labeling of proteins with radioisotopes are known in theart. For example, such methods are found in Ohta et al., Molec. Cell3:535-541 (1999), which is hereby incorporated by reference in itsentirety.

Production of proteins having tags by recombinant means is well known,and kits for producing such proteins are commercially available. Forexample, such a kit and its use is described in the QIAexpress Handbookfrom Qiagen by Joanne Crowe et al., hereby expressly incorporated byreference.

The functionalization of labels with chemically reactive groups such asthiols, amines, carboxyls, etc. is generally known in the art. In apreferred embodiment, the tag is functionalized to facilitate covalentattachment. The covalent attachment of the tag may be either direct orvia a linker. In one embodiment, the linker is a relatively shortcoupling moiety, which is used to attach the molecules. A couplingmoiety may be synthesized directly onto a component of the invention andcontains at least one functional group to facilitate attachment of thetag. Alternatively, the coupling moiety may have at least two functionalgroups, which are used to attach a functionalized component to afunctionalized tag, for example. In an additional embodiment, the linkeris a polymer. In this embodiment, covalent attachment is accomplishedeither directly, or through the use of coupling moieties from thecomponent or tag to the polymer. In a preferred embodiment, the covalentattachment is direct, that is, no linker is used. In this embodiment,the component preferably contains a functional group such as acarboxylic acid that is used for direct attachment to the functionalizedtag. It should be understood that the component and tag may be attachedin a variety of ways, including those listed above. In a preferredembodiment, the tag is attached to the amino or carboxl terminus of thepolypeptide. As will be appreciated by those in the art, the abovedescription of the covalent attachment of a label applies to theattachment of virtually any two molecules of the present disclosure.

In a preferred embodiment, the tag is functionalized to facilitatecovalent attachment, as is generally outlined above. Thus, a widevariety of tags are commercially available which contain functionalgroups, including, but not limited to, isothiocyanate groups, aminogroups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonylhalides, all of which may be used to covalently attach the tag to asecond molecule, as is described herein. The choice of the functionalgroup of the tag will depend on the site of attachment to either alinker, as outlined above or a component of the invention. Thus, forexample, for direct linkage to a carboxylic acid group of a protein,amino modified or hydrazine modified tags will be used for coupling viacarbodiimide chemistry, for example using1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) as is known in theart (see Set 9 and Set 11 of the Molecular Probes Catalog, supra; seealso the Pierce 1994 Catalog and Handbook, pages T-155 to T-200, both ofwhich are hereby incorporated by reference). In one embodiment, thecarbodiimide is first attached to the tag, such as is commerciallyavailable for many of the tags described herein.

Alternative Activation State Indicators

An alternative activation state indicator useful with the instantinvention is one that allows for the detection of activation byindicating the result of such activation. For example, phosphorylationof a substrate can be used to detect the activation of the kinaseresponsible for phosphorylating that substrate. Similarly, cleavage of asubstrate can be used as an indicator of the activation of a proteaseresponsible for such cleavage. Methods are well known in the art thatallow coupling of such indications to detectable signals, such as thelabels and tags described above in connection with binding elements. Forexample, cleavage of a substrate can result in the removal of aquenching moiety and thus allowing for a detectable signal beingproduced from a previously quenched label.

FACS Analysis

In a preferred embodiment, the present invention provides methods fordetermining an activatable element's activation profile for a singlecell. The methods comprise sorting cells by FACS on the basis of theactivation state of at least two activatable elements. BEs (e.g.activation state-specific antibodies) are used to sort cells on thebasis of activatable element activation state, and can be detected asdescribed below. Alternatively, non-BE systems as described above can beused in any system described herein.

When using fluorescent labeled components in the methods andcompositions of the present invention, it will recognized that differenttypes of fluorescent monitoring systems, e.g., FACS systems, can be usedto practice the invention. Preferably, FACS systems are used or systemsdedicated to high throughput screening, e.g 96 well or greatermicrotiter plates. Methods of performing assays on fluorescent materialsare well known in the art and are described in, e.g., Lakowicz, J. R.,Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983);Herman, B., Resonance energy transfer microscopy, in: FluorescenceMicroscopy of Living Cells in Culture, Part B, Methods in Cell Biology,vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press(1989), pp. 219-243; Turro, N.J., Modern Molecular Photochemistry, MenloPark: Benjamin/Cummings Publishing Col, Inc. (1978), pp. 296-361.

Fluorescence in a sample can be measured using a fluorimeter. Ingeneral, excitation radiation, from an excitation source having a firstwavelength, passes through excitation optics. The excitation opticscause the excitation radiation to excite the sample. In response,fluorescent proteins in the sample emit radiation that has a wavelengththat is different from the excitation wavelength. Collection optics thencollect the emission from the sample. The device can include atemperature controller to maintain the sample at a specific temperaturewhile it is being scanned. According to one embodiment, a multi-axistranslation stage moves a microtiter plate holding a plurality ofsamples in order to position different wells to be exposed. Themulti-axis translation stage, temperature controller, auto-focusingfeature, and electronics associated with imaging and data collection canbe managed by an appropriately programmed digital computer. The computeralso can transform the data collected during the assay into anotherformat for presentation. In general, known robotic systems andcomponents can be used.

In a preferred embodiment, flow cytometry is used to detectfluorescence. Other methods of detecting fluorescence may also be used,e.g., Quantum dot methods (see, e.g., Goldman et al., J. Am. Chem. Soc.(2002) 124:6378-82; Pathak et al. J. Am. Chem. Soc. (2001) 123:4103-4;and Remade et al., Proc. Natl. Sci. USA (2000) 18:553-8, each expresslyincorporated herein by reference) as well as confocal microscopy. Ingeneral, flow cytometry involves the passage of individual cells throughthe path of a laser beam. The scattering the beam and excitation of anyfluorescent molecules attached to, or found within, the cell is detectedby photomultiplier tubes to create a readable output, e.g. size,granularity, or fluorescent intensity.

The detecting, sorting, or isolating step of the methods of the presentinvention can entail fluorescence-activated cell sorting (FACS)techniques, where FACS is used to select cells from the populationcontaining a particular surface marker, or the selection step can entailthe use of magnetically responsive particles as retrievable supports fortarget cell capture and/or background removal. A variety of FACS systemsare known in the art and can be used in the methods of the invention(see e.g., WO99/54494, filed Apr. 16, 1999; U.S.S.N. 20010006787, filedJul. 5, 2001, each expressly incorporated herein by reference).

In a preferred embodiment, a FACS cell sorter (e.g. a FACSVantage™ CellSorter, Becton Dickinson Immunocytometry Systems, San Jose, Calif.) isused to sort and collect cells based on their activation profile(positive cells) in the presence or absence of potentiation.

In one embodiment, the cells are first contacted withfluorescent-labeled activation state-specific BEs (e.g. antibodies)directed against specific isoforms of specific activatable elements. Insuch an embodiment, the amount of bound BE on each cell can be measuredby passing droplets containing the cells through the cell sorter. Byimparting an electromagnetic charge to droplets containing the positivecells, the cells can be separated from other cells. The positivelyselected cells can then be harvested in sterile collection vessels.These cell-sorting procedures are described in detail, for example, inthe FACSVantage™ Training Manual, with particular reference to sections3-11 to 3-28 and 10-1 to 10-17, which is hereby incorporated byreference in its entirety.

In another embodiment, positive cells can be sorted using magneticseparation of cells based on the presence of an isoform of anactivatable element. In such separation techniques, cells to bepositively selected are first contacted with specific binding element(e.g., an antibody or reagent that binds an isoform of an activatableelement). The cells are then contacted with retrievable particles (e.g.,magnetically responsive particles) that are coupled with a reagent thatbinds the specific element. The cell-binding element-particle complexcan then be physically separated from non-positive or non-labeled cells,for example, using a magnetic field. When using magnetically responsiveparticles, the positive or labeled cells can be retained in a containerusing a magnetic filed while the negative cells are removed. These andsimilar separation procedures are described, for example, in the BaxterImmunotherapy Isolex training manual which is hereby incorporated in itsentirety.

In a preferred embodiment, methods for the determination of a receptorelement activation state profile for a single cell are provided. Themethods comprise providing a population of cells and sorting thepopulation of cells by FACS. Preferably, cells are separated on thebasis of the activation state of at least two activatable elements. In apreferred embodiment, a multiplicity of activatable elementactivation-state antibodies are used to simultaneously determine theactivation state of a multiplicity of elements.

In a preferred embodiment, cell sorting by FACS on the basis of theactivation state of at least two elements is combined with adetermination of other FACS readable outputs, such as the presence ofsurface markers, granularity and cell size to provide a correlationbetween the activation state of a multiplicity of elements and othercell qualities measurable by FACS for single cells.

In a preferred embodiment cell sorting is done on the basis of elementclustering. In one embodiment element clustering can be identified usinga doublet discriminator.

A doublet discriminator is so named because of its initial use indiscriminating a doublet of G0 or G1 stage cells from a single G2 or Mstage cell. As is well known in the art, cells in the G0 and G1 stagesof the cell cycle have a single complement of DNA, while cells in the G2and M stages have doubled their DNA complement in anticipation of celldivision. Accordingly, a FACS machine only detecting the amount of DNAwithin a cell would not be able to discriminate between a doublet of G0or G1 stage cells, two cells containing a single complement of DNA each,and a G2 or M stage cell, a single cell containing a double complementof DNA. However, because a doublet discriminator also incorporates cellsize detection, as discussed below, the doublet can discriminated fromthe single G2 or M stage cell as being twice the size, though having thesame amount of DNA.

As used herein a doublet discriminator functions by comparing two FACSreadable outputs. The first of these two FACS readable outputs isfluorescent intensity. In general, a FACS machine will use a laser toexcite fluorochromes and make use of photomultiplier tubes to detect thephotons emitted by these fluorochromes. The emitted photons are thenconverted to a voltage reading, termed the voltage pulse distribution.Fluorescent intensity can be measured either as the maximum intensity ofthe voltage pulse distribution or by integrating the voltage pulsedistribution, rather than only taking the maximum, to give a moreprecise value. The second FACS readable output necessary for doubletdiscrimination is signal pulse width. Signal pulse width is ameasurement of the time taken for the cell to pass through the laserbeam and is commonly termed the “Time of Flight” (TOF). As the TOF isproportional to the size of the cell passing through the laser, withlarger cells taking longer time periods to pass through the beam andsmaller cells taking shorter time periods, the TOF is also proportionalto the surface area of that cell.

The doublet discriminator is effective in detecting element clusteringdue to the proportionality between signal pulse width and surface area.By normalizing the fluorescent intensity over the cell's surface area,clustering is detected as an increase in intensity per unit of surfacearea as compared to intensity in a non-clustered state. This normalizedfluorescent intensity is then plotted over time to represent an increaseor decrease in clustering after a certain event, such as administrationof a bioactive agent. An example of detecting clustering using a doubletdiscriminator is presented below in Example 1.

As will be appreciated, the present invention also provides for theordering of element clustering events in signal transduction.Particularly, the present invention allows the artisan to construct anelement clustering and activation hierarchy based on the correlation oflevels of clustering and activation of a multiplicity of elements withinsingle cells. Ordering can be accomplished by comparing the activationstate of a cell or cell population with a control at a single timepoint, or by comparing cells at multiple time points to observesubpopulations arising out of the others.

The present invention provides a valuable method of determining thepresence of cellular subsets within cellular populations. Ideally,signal transduction pathways are evaluated in homogeneous cellpopulations to ensure that variances in signaling between cells do notqualitatively nor quantitatively mask signal transduction events andalterations therein. As the ultimate homogeneous system is the singlecell, the present invention allows the individual evaluation of cells toallow true differences to be identified in a significant way.

Thus, the invention provides methods of distinguishing cellular subsetswithin a larger cellular population. As outlined herein, these cellularsubsets often exhibit altered biological characteristics (e.g.activation states, altered response to potentiation) as compared toother subsets within the population. For example, as outlined herein,the methods of the invention allow the identification of subsets ofcells from a population such as primary cell populations, e.g.peripheral blood mononuclear cells, that exhibit altered responses (e.g.drug resistance or susceptibility) as compared to other subsets. Inaddition, this type of evaluation distinguishes between differentactivation states, altered responses to potentiation, cell lineages,cell differentiation states, etc.

As will be appreciated, these methods provide for the identification ofdistinct signaling cascades for both artificial and stimulatoryconditions in complex cell populations, such a peripheral bloodmononuclear cells, or naive and memory lymphocytes.

Additional Techniques

As will be appreciated by one of skill in the art, the instant methodsand compositions find use in a variety of other assay formats inaddition to FACS analysis. For example, a chip analogous to a DNA chipcan be used in the methods of the present invention. Arrayers andmethods for spotting nucleic acid to a chip in a prefigured array areknown. In addition, protein chips and methods for synthesis are known.These methods and materials may be adapted for the purpose of affixingactivation state BEs to a chip in a prefigured array. In a preferredembodiment, such a chip comprises a multiplicity of element activationstate BEs, and is used to determine an element activation state profilefor elements present on the surface of a cell.

In an alternative embodiment, a chip comprises a multiplicity of the“second set BEs,” in this case generally unlabeled. Such a chip iscontacted with sample, preferably cell extract, and a secondmultiplicity of BEs comprising element activation state specific BEs isused in the sandwich assay to simultaneously determine the presence of amultiplicity of activated elements in sample. Preferably, each of themultiplicity of activation state-specific BEs is uniquely labeled tofacilitate detection.

In an alternative embodiment confocal microscopy can be used to detectactivation profiles for individual cells. Confocal microscopy relies onthe serial collection of light from spatially filtered individualspecimen points, which is then electronically processed to render amagnified image of the specimen. The signal processing involved confocalmicroscopy has the additional capability of detecting labeled bindingelements within single cells, accordingly in this embodiment the cellscan be labeled with one or more binding elements. In preferredembodiments the binding elements used in connection with confocalmicroscopy are antibodies conjugated to fluorescent labels, howeverother binding elements, such as other proteins or nucleic acids are alsopossible.

In an additional embodiment the methods and compostitions of the instantinvention can be used in conjunction with an “In-Cell Western Assay.” Insuch an assay, cells are initially grown in standard tissue cultureflasks using standard tissue culture techniques. Once grown to optiumconfluency, the growth media is removed and cells are washed andtrypsinized. The cells can then be counted and volumes sufficient totransfer the appropriate number of cells are aliquoted into microwellplates (e.g., Nunc™ 96 Microwell™ plates). The individual wells are thengrown to optimum confluency in complete media whereupon the media isreplaced with serum-free media. At this point controls are untouched,but experimental wells are incubated with a bioactive agent, e.g. EGF.After incubation with the bioactive agent cells are fixed and stainedwith labeled antibodies to the actiavation elements being investigated.Once the cells are labeled, the plates can be scanned using an imagersuch as the Odyssey Imager (LiCor, Lincoln Nebr.) using techniquesdescribed in the Odyssey Operator's Manual v1.2., which is herebyincorporated in its entirety. Data obtained by scanning of the multiwellplate can be analysed and activation profiles determined as describedbelow.

Types of Bioactive Candidates that can be Used

In a preferred embodiment, the invention provides methods for screeningfor a bioactive agent capable of modulating element activity. Forexample, drugs and drug candidates can be screened to either evaluatethe effect of the drug/candidate on activation profiles, or to createdesired profiles. The methods comprise contacting a cell with acandidate bioactive agent and determining element activation in saidcell using the techniques outlined herein, including FACS.

In a preferred embodiment, the method comprises contacting a pluralityof cells with a plurality of candidate bioactive agents and sorting thecells by FACS on the basis of the activation of at least one element.

By “candidate bioactive agent”, “candidate agent”, “candidatemodulator”, “candidate modulating agent”, or “exogeneous compound” orgrammatical equivalents herein is meant any molecule, e.g., protein,small organic molecule, carbohydrates (including polysaccharides),polynucleotide, lipids, etc. Specifically included in the definition ofcandidate bioactive agent are drugs. Generally a plurality of assaymixtures can be run in parallel with different agent concentrations toobtain a differential response to the various concentrations. Typically,one of these concentrations can serve as a negative control, i.e., atzero concentration or below the level of detection. In addition,positive controls can be used.

Candidate agents encompass numerous chemical classes. In a preferredembodiment, the candidate agents are small molecules. In anotherpreferred embodiment, the candidate agents are organic molecules,particularly small organic molecules, comprising functional groupsnecessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups. The candidate agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more chemical functional groups.

Candidate agents are obtained from a wide variety of sources, as will beappreciated by those in the art, including libraries of synthetic ornatural compounds. As will be appreciated by those in the art, thepresent invention provides a rapid and easy method for screening anylibrary of candidate modulators, including the wide variety of knowncombinatorial chemistry-type libraries.

In a preferred embodiment, candidate agents are synthetic compounds, asdescribed above in connection with binding elements. One advantage ofthe present method is that it is not necessary to characterize thecandidate agent prior to the assay. Using the methods of the presentinvention, any candidate agents can be screened for the ability tomodulate (e.g., increase or decease) the activity of an activatableelement. In addition, as is known in the art, coding tags using splitsynthesis reactions may be used to essentially identify the chemicalmoieties tested.

Alternatively, a preferred embodiment utilizes libraries of naturalcompounds, as candidate agents, in the form of bacterial, fungal, plantand animal extracts that are available or readily produced.

Additionally, natural or synthetically produced libraries and compoundsare readily modified through conventional chemical, physical andbiochemical means. Known pharmacological agents may be subjected todirected or random chemical modifications, including enzymaticmodifications, to produce structural analogs.

In a preferred embodiment, candidate agents include proteins, nucleicacids, and chemical moieties.

In a preferred embodiment, the candidate agents are proteins, as definedabove. In a preferred embodiment, the candidate agents are naturallyoccurring proteins or fragments of naturally occurring proteins. Thus,for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be tested, asis more fully described below. In this way libraries of prokaryotic andeukaryotic proteins may be made for screening against any number ofcandidate agents. Particularly preferred in this embodiment arelibraries of bacterial, fungal, viral, and mammalian proteins, with thelatter being preferred, and human proteins being especially preferred.

In a preferred embodiment, the candidate agents are peptides of fromabout 2 to about 50 amino acids, with from about 5 to about 30 aminoacids being preferred, and from about 8 to about 20 being particularlypreferred. The peptides may be digests of naturally occurring proteinsas is outlined above, random peptides, or “biased” random peptides. By“randomized” or grammatical equivalents herein is meant that eachnucleic acid and peptide consists of essentially random nucleotides andamino acids, respectively. Since generally these random peptides (ornucleic acids, discussed below) are chemically synthesized, they mayincorporate any nucleotide or amino acid at any position. The syntheticprocess can be designed to generate randomized proteins or nucleicacids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized candidate bioactive proteinaceous agents.

The library should provide a sufficiently structurally diversepopulation of randomized agents to effect a probabilistically sufficientrange of diversity to allow interaction with a particular activatableprotein. Accordingly, an interaction library must be large enough sothat at least one of its members will have a structure that interactswith an activatable protein or other specific components of the signaltransduction pathway involving the activatable protein. Although it isdifficult to gauge the required absolute size of an interaction library,nature provides a hint with the immune response: a diversity of 10⁷-10⁸different antibodies provides at least one combination with sufficientaffinity to interact with most potential antigens faced by an organism.Published in vitro selection techniques have also shown that a librarysize of 10⁷ to 10⁸ is sufficient to find structures with affinity for atarget. A library of all combinations of a peptide 7 to 20 amino acidsin length, such as generally proposed herein, has the potential to codefor 20⁷ (10⁹) to 20²⁰. Thus, with libraries of 10⁷ to 10⁸ differentmolecules the present methods allow a “working” subset of atheoretically complete interaction library for 7 amino acids, and asubset of shapes for the 20²⁰ library. Thus, in a preferred embodiment,at least 10⁶, preferably at least 10⁷, more preferably at least 10⁸ andmost preferably at least 10⁹ different sequences are simultaneouslyanalyzed in the subject methods. Preferred methods maximize library sizeand diversity.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation ofcysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In a preferred embodiment, the bias is towards peptides or nucleic acidsthat interact with known classes of molecules. For example, when thecandidate agent is a peptide, it is known that much of intracellularsignaling is carried out via short regions of polypeptides interactingwith other polypeptides through small peptide domains. For instance, ashort region from the HIV-1 envelope cytoplasmic domain has beenpreviously shown to block the action of cellular calmodulin. Regions ofthe Fas cytoplasmic domain, which shows homology to the mastoparan toxinfrom Wasps, can be limited to a short peptide region with death-inducingapoptotic or G protein inducing functions. Magainin, a natural peptidederived from Xenopus, can have potent anti-tumor and anti-microbialactivity. Short peptide fragments of a protein kinase C isozyme (βPKC),have been shown to block nuclear translocation of βPKC in Xenopusoocytes following stimulation. And, short SH-3 target peptides have beenused as psuedosubstrates for specific binding to SH-3 proteins. This isof course a short list of available peptides with biological activity,as the literature is dense in this area. Thus, there is much precedentfor the potential of small peptides to have activity on intracellularsignaling cascades. In addition, agonists and antagonists of any numberof molecules may be used as the basis of biased randomization ofcandidate modulators as well.

Thus, a number of molecules or protein domains are suitable as startingpoints for the generation of biased randomized candidate modulators. Alarge number of small molecule domains are known, that confer a commonfunction, structure or affinity. In addition, as is appreciated in theart, areas of weak amino acid homology may have strong structuralhomology A number of these molecules, domains, and/or correspondingconsensus sequences, are known, including, but are not limited to, SH-2domains, SH-3 domains, Pleckstrin, death domains, proteasecleavage/recognition sites, enzyme inhibitors, enzyme substrates, andTraf.

In a preferred embodiment, the candidate modulating agent is apolypeptide. In another preferred embodiment, the polypeptide is acyclic peptide having at least 4 to 20 amino acids. Also in anotherpreferred embodiment, the polypeptide is a catalytically inactivepolypeptide. Examples of catalytically inactive polypeptides include,but are not limited to, catalytically inactive activatable proteins and,more specifically a catalytically inactive kinases (e.g., PI3K) orcaspases. In a further aspect, the candidate modulating agent is peptidefragment of an activatable protein, wherein the peptide fragmentcomprises an amino acid sequence that is a subsequence of thefull-length amino acid sequence of the activatable protein.

In a preferred embodiment, the candidate agents are nucleic acids asdescribed above in connection with binding elements.

In a preferred embodiment, the candidate agents are organic moieties asdescribed above in connection with binding elements.

As will be appreciated by those in the art, it is possible to screenmore than one type of candidate agent at a time, e.g., by combining thecandidate agents in the methods of the present invention. Thus, thelibrary of candidate agents used may include only one type of agent(i.e. peptides), or multiple types (peptides and organic agents).

General Screening Methods

In a generalized protocol for determining an activation profile, cellsare initially suspended in a small volume of media. The cells can thenbe aliquoted into a multiwell plate or other appropriate substrate andcan be incubated in the presence or absence of a bioactive agent. Afterincubation is allowed to continue for an appropriate time and at anappropriate temperature the incubation can be terminated and the cellscan be fixed as is well known in the art.

Once fixed, the cells can be pelleted and resuspended in methanol topermeabilize, although other methods of permeabilization are alsocompatible with the instant invention. Cells can be stored at this pointor combined with labeled binding elements and analyzed right away.

By “combined” is meant the combining of the various components in areaction mixture in vitro or in a cell in vivo under conditions whichpromote an activity that is detectable using known methods or using themethods of the present invention (e.g., the binding of an antibody to acorresponding antigen or isoform of an activatable protein, oractivation state of an activatable protein).

Analysis begins by creating a response panel. The response panel isapproximated in a two dimensional array of nodes (in rows) and states(in columns), similar to the experimental layout of the multiwell plate.The nodes correspond to each of the proteins being studied in theprofile. Other response panels having other characteristics can alsofind use in the instant invention. The response panels represent theFACS (or other detection method) output for each sample from themultiwell plate. Typically, the log 2 of [MFI stimulated/MFIunstimulated] is taken, however other normalization methodologies areknown and can be used in conjunction with the instant invention. Forbasal nodes, the log 2 of [MFI basal x/MFI sample minimum basal] can beused to place measurements on the same scale. In order to identify anactivation profile, the node states for all samples are collected andthe variance across samples is studied. When multiple timepoints areincluded, each timepoint of a sample can be treated as a separatesample. Those node states with variance greater than that seen in anormal cell are typically included in the activation profile.

As discussed above, the instant invention provides methods andcompositions for the detection of the activation of elements in cells.As used herein the term cells and grammatical equivalents herein inmeant any cell, preferably any prokaryotic or eukaryotic cell.

Suitable prokaryotic cells include, but are not limited to, bacteriasuch as E. coli, various Bacillus species, and the extremophile bacteriasuch as thermophiles, etc.

Suitable eukaryotic cells include, but are not limited to, fungi such asyeast and filamentous fungi, including species of Aspergillus,Trichoderma, and Neurospora; plant cells including those of corn,sorghum, tobacco, canola, soybean, cotton, tomato, potato, alfalfa,sunflower, etc.; and animal cells, including fish, birds and mammals.Suitable fish cells include, but are not limited to, those from speciesof salmon, trout, tulapia, tuna, carp, flounder, halibut, swordfish, codand zebrafish. Suitable bird cells include, but are not limited to,those of chickens, ducks, quail, pheasants and turkeys, and other junglefoul or game birds. Suitable mammalian cells include, but are notlimited to, cells from horses, cows, buffalo, deer, sheep, rabbits,rodents such as mice, rats, hamsters and guinea pigs, goats, pigs,primates, marine mammals including dolphins and whales, as well as celllines, such as human cell lines of any tissue or stem cell type, andstem cells, including pluripotent and non-pluripotent, and non-humanzygotes.

Suitable cells also include those cell types implicated in a widevariety of disease conditions, even while in a non-diseased state.Accordingly, suitable eukaryotic cell types include, but are not limitedto, tumor cells of all types (particularly melanoma, myeloid leukemia,carcinomas of the lung, breast, ovaries, colon, kidney, prostate,pancreas and testes), cardiomyocytes, dendritic cells, endothelialcells, epithelial cells, lymphocytes (T-cell and B cell), mast cells,eosinophils, vascular intimal cells, macrophages, natural killer cells,erythrocytes, hepatocytes, leukocytes including mononuclear leukocytes,stem cells such as haemopoetic, neural, skin, lung, kidney, liver andmyocyte stem cells (for use in screening for differentiation andde-differentiation factors), osteoclasts, chondrocytes and otherconnective tissue cells, keratinocytes, melanocytes, liver cells, kidneycells, and adipocytes. Particularly preferred are primary disease statecells, such as primary tumor cells. Suitable cells also include knownresearch cells, including, but not limited to, Jurkat T cells, NIH3T3cells, CHO, COS, etc. See the ATCC cell line catalog, hereby expresslyincorporated by reference.

In a preferred embodiment the cells used in the present invention aretaken from a patient. As used herein “patient” refers to both human andother animals as well as other organisms, such as experimental animals.Thus the methods and compositions are applicable to both human andveterinary applications. In a preferred embodiment the patient is amammal, and in a more preferred embodiment the patient is human.

Screening of Agents in the Potentiated Model

In a generalized protocol for study of potentiation, cells (e.g.including but not limited to any cell type described above in connectionwith the general screening methods) are initially suspended in a smallvolume of media and counted. An appropriate amount of viable cells fromeach should be present in the sample (for four core stains), plus anappropriate amount for each stored sample. Cells are then diluted to anappropriate concentration, and an appropriate volume of each isaliquoted into a multiwell plate or other appropriate substrate, asshown below (See Example 3). Potentiators are then added an appropriateconcentration and volume into each column of cells. Potentiation isallowed to continue for an appropriate time and at an appropriatetemperature. To end potentiation, cells can be washed to remove thepotentiator. Potentiatied cells can then be incubated with a bioactiveagent in order to screen the effects of that agent. Exposure to theagent can be terminated at various time points by fixing the cells as isknown in the art.

After potentiation, the cells can be pelleted and resuspended inmethanol to permeabilize, although as discussed above, other methods ofpermeabilization are also compatible with the instant invention. Cellscan be stored at this point or incubated with labeled binding elementsand analyzed right away.

Analysis proceeds as described above, including the creation of aresponse panel, normalization of the nodes, and variances calculated.

Once the nodes have been normalized and the variances determined,unsupervised clustering of biosignature node states is used to groupsamples. Distribution of clinical parameters in resulting groups isdetermined using statistical significance testing (e.g. Chi Squared andStudent's t-test on previously defined hypotheses). Groups of sampleswith similar potentiation can then be given designations and pathwaymaps, based on all possible pathway interactions observed, can behighlighted and dimmed according to observations of each group. Ingeneral, if more than half the samples in the group display potentiationabove the median for all samples, then that pathway is highlighted.Otherwise, the pathway is dimmed.

Pathway maps can be built out for individual samples based on theirobserved potentiation, and based on these maps a sample can bephenotyped as belonging to a group (and will be predicted to havesimilar characteristics as that group). Once a predictive panel for ismade for a group, it can be validated on an additional set of samplessimilar to the original. With the additional data, the map can beminimized and optimized to be as accurate as possible with as few aspossible node state measurements for a given sample.

Analysis

Advances in flow cytometry have enabled the individual cell enumerationof up to thirteen simultaneous parameters (De Rosa et al., 2001) and aremoving towards the study of genomic and proteomic data subsets (Krutzikand Nolan, 2003; Perez and Nolan, 2002). As the number of parameters,epitopes, and samples have increased, the complexity of flow cytometryexperiments and the challenges of data analysis have grown rapidly. Anadditional layer of data complexity has been added by the development ofstimulation panels which enable the study of signal transduction nodesunder a growing set of experimental conditions. In order to deal withthe resulting experimental and informatics challenges, the instantinvention provies techniques of arraying flow cytometry experiments andapproximating the results as fold changes using a heat map to facilitateevaluation. There are a large number of useful ways that flow cytometryexperiments can be displayed, such as arraying. Generally speaking,arrayed flow cytometry experiments simplify multidimensional flowcytometry data based on experimental design and observed differencesbetween flow cytometry samples. One common way of comparing changes in aset of flow cytometry samples is to overlay histograms of one parameteron the same plot. Arrayed flow cytometry experiments ideally contain acontrol population of unstained, control stained, or unstimulated cellsagainst which experimental samples are compared. This control is placedin the first position of the array, and subsequent experimental samplesfollow the control in the sequence. However, large numbers of histogramsor sets of 2D flow cytometry plots can be unwieldy.

Differences between experimental samples can be highlighted by coloringthe peaks relative to the change in median fluorescence index (MFI) ofeach sample. The change from this basal state is calculated as:

[Equation  1-1, change  of  node  state  from  basal]$x^{\prime}{\log_{2}\left( \frac{x_{experimental}}{x_{basal}} \right)}$

In Equation 1-1, the fold change for a parameter (e.g., proteinphosphorylation, protein expression level) is represented by the changebetween an experimental, stimulated (e.g. potentiated) MFI and a controlMFI, such as the MFI of the same cells without stimulation. Because thelog₂ of the change is taken, increases are positive values, decreasesare negative values, and small changes are close to zero. To clearlyconvey the results of an experiment, the same color values can bearrayed according to the experimental design. In an experiment of thisrelatively small size, the advantage of an arrayed approach is lessobvious, and it is the expansion of this technique to a large number oftargets, samples, timepoints, and cell subsets that has driven thedevelopment of arrayed flow cytometry.

A specific example is useful in describing these aspects of the instantinvention. The target phospho-protein in this example, Stat1, is termeda signaling node, and the stimulation conditions under which it isstudied are termed node states. This general language is useful forarrayed flow cytometry experiments because it describes diverse cellsignaling mechanisms. Thus, mechanisms by which intracellular signalstraverse the cell, including phosphorylation, proteolytic cleavage,ubiquitinylation, acetylation, expression level and otherpost-translational protein states. Flow cytometry assays are describedto monitor each of these events and thus numerous proteomic events canbe mapped simultaneously in single cells.

Arraying several signal transduction nodes under a common set ofconditions, which results in a response panel, provides a twodimensional profile of a target cell population. In this particularexample the signaling of six phospho-proteins in the U937 cell line arecompared. These phospho-proteins can be assayed simultaneously inindividual cells or assayed sequentially in replicate samples. In thisway every channel of multiparameter data can be compared simultaneouslyand samples analyzed in parallel can be combined and compared. Thistechnique is especially useful when moving a well-developed series ofassays into a model where each sample will be profiled uniformly (e.g.,patient samples). By subtracting the responses in one panel from anothersample, or a universal control, a researcher can quickly identify theremaining signaling mechanisms that differ between the two samples.

Arrayed flow cytometry can also be used to screen individual or combinedstimulations for a fixed set of signaling nodes. In a plate-basedarrayed flow cytometry experiment, the rows and columns represent a gridof stimulation conditions being tested for a response. Each well in aplate might contain the same cells which, as a population, function as asignal transduction test tube. Stimuli are given at x, y coordinatesand, following a set time of stimulation, the cells are fixed and eachset of cells stained simultaneously for the same set of multipletargets. The natural formatting of arrayed flow cytometry experiments in96-well plates carries several advantages. Equipment allowingmultichannel addition of reagents lowers experimental error, enforcesuniformity, and makes it possible to start and stop in tandem a largenumber of biochemical experiments with short time frames.

In another example of arrayed flow cytometry, signaling mechanisms inperipheral blood leukocytes (PBL) are compared at four minute timepointsfollowing potentiation with phorbol 12-myristate 13-acetate(PMA)/ionomycin. Cells were stained with a surface marker for myeloidlineage cells (CD33) and phosphorylation of four signaling proteins(p38, Stat1, and p53). Signaling was initiated sequentially in a reversetimecourse, and all samples were fixed and stained in unison.Particularly striking in this experiment is that the response of somephospho-proteins is only revealed when a subset of cells is analyzedseparately from the background of quiescent non-responders. This resultwould have been extremely difficult to obtain using classical,mono-parameter technologies for analysis of phospho-proteins (e.g.,Western blotting), and would have likely involved physical isolation ofmyeloid cells from total PBL, a process which can disrupt intracellularsignaling.

In order to clarify the differences between experimental samples andcontrols, MFI changes are approximated as a log₂ fold change.Enumerating signal transduction events in this way makes flow cytometrydata portable to statistical methods and clustering algorithms. (Notethat the use of the term “clustering” in this context is different fromreceptor clustering; this context refers to algorithms that cluster datapoints together in any number of ways. Additionally, cross-platformcomparison of samples can be performed when diverse parameters areplaced on a similar fold induction scale (e.g., protein expression, RNAcontent, DNA content and cell cycle representation, proteinphosphorylation under diverse conditions, length of repeated geneticelements, patient survival time, cell death following ex vivostimulation).

Once a vital set of parameters is identified, unsupervised clustering ofsamples with similar profiles provides a powerful way to identify groupsand relationships within a population of samples. According toconventions developed by DNA microarray biologists, populations fromflow cytometry can be arranged where each cell or sample of interest isplaced in a column and each parameter of interest is placed in a row,analogous to experiments and genes. Once the appropriate populations andparameters have been entered, a number of clustering techniques can bedone. Generally, successful use of unsupervised clustering relies onparameters of interest being intelligently chosen, or selected using analgorithm as with variance mapping (see below), such that most or allfactors being clustered are important to the group of samples beingclustered. This is because unsupervised clustering groups samples solelybased on similarities in the values reported for each signaling nodestate. This technique carries the risk that the groups identified thisway might not be clinically relevant, but provides a strong incentivebecause potentially artificial, pre-organized groups are avoided. Asoutlined below this approach was used to identify signaling pathologiesin acute myeloid leukemia. Compounds with specific activity on a fixedsystem can also be identified easily using this approach.

Prior to clustering of flow cytometry data, the populations of interestand the method for characterizing these populations are determined. Inarrayed flow cytometry there are at least two general ways ofidentifying populations for clustering:

-   -   1. “Outside-in” comparison of parameter sets for individual        samples or subset (e.g. patients in a trial). In this more        common case, cell populations are homogenous or lineage gated in        such a way as to create distinct sets considered to be        homogenous for targets of interest. An example of sample-level        comparison would be the identification of signaling profiles in        the tumor cells of a patient and correlation of these profiles        with non-random distribution of clinical responses. This is        considered an outside-in approach because the population of        interest is pre-defined prior to the mapping and comparison of        its profile to other populations.    -   2. “Inside-out” comparison of parameters at the level of        individual cells in a heterogeneous population. An example of        this would be the signal transduction state mapping of mixed        hematopoietic cells under certain conditions and subsequent        comparison of computationally identified cell clusters with        lineage specific markers. This could be considered an inside-out        approach to single cell studies as it does not presume the        existence of specific populations prior to classification. A        major drawback of this approach is that it creates populations        which, at least initially, require multiple transient markers to        enumerate and may never be accessible with a single cell surface        epitope. As a result, the biological significance of such        populations can be difficult to determine. The main advantage of        this unconventional approach is the unbiased tracking of cell        populations without drawing potentially arbitrary distinctions        between lineages or cell types.

Each of these techniques capitalizes on the ability of flow cytometry todeliver large amounts of multiparameter data at the single cell level.For cancer, a third “meta-level” of tumor data exists because tumors aregenerally treated as a single entity and classified according tohistorical techniques. These techniques have included organ or tissue oforigin, degree of differentiation, proliferation index, metastaticspread, and genetic or metabolic data regarding the patient.

In addition, the present invention provides variance mapping techniquesfor mapping tumor signalling space. These methods represent asignificant advance in the study of tumor biology because it enablescomparison of tumors independent of a putative normal control.Traditional differential state analysis methods (e.g., DNA microarrays,subtractive Northern blotting) generally rely on the comparison oftumors from each patient sample with a normal control, generallyadjacent and theoretically untransformed tissue. Alternatively, theyrely on multiple clusterings and reclusterings to group and then furtherstratify patient samples according to phenotype. In contrast, variancemapping of tumor states compares tumor samples first with themselves andthen against the parent tumor population. As a result, node states withthe most diversity among tumors provide the core parameters in thedifferential state analysis. Given a pool of diverse tumors, thistechnique allows a researcher to identify the molecular events thatunderlie differential tumor pathology (e.g., tumor responses tochemotherapy), as opposed to differences between tumors and a proposednormal control.

The variance (σ²) is one way can be represented the diversity of statesobserved for a node across a set of tumor samples. For arrayed flowcytometry experiments the variance of a node state is calculated as theaverage squared deviation of each sample's measurement from the mean ofthe sample set:

                   [Equation  1-2, variance  of  a  node  state]$\sigma^{2} = \frac{\sum\;\left( {x - \overset{\_}{x}} \right)^{2}}{N}$

In Equation 1-2, x is each sample's log₂ fold node state measurement, xis the average of x across all samples, and N is the number of samples.Parameters where node state measurements vary frequently and by largeamounts will have the highest variance. One area for refinement usingthis technique is in the comparison of diverse parameters that may notalways report similar differences in MFI for technical reasons. The MFIshift for a node state can be lowered due to poor fluorophore labelingof a BE or high BE staining background. These issues generally reducethe variance of a node state, causing potentially significantinformation to be overlooked. In performing clinical profiling of nodestates, it is important to work with well characterized reagents and tocompare nodes and their states on similar scales.

When variance mapping is used to profile the signaling space of patientsamples, tumors whose signaling is perturbed in similar ways are groupedtogether, regardless of tissue or cell type of origin (see profiling ofAML). Similarly, two tumors that are thought to be relatively alikebased on lineage markers or tissue of origin could have vastly differentabilities to interpret environmental stimuli and would be profiled intwo different groups. What we have found with this technique is that thepotentiated signaling of acute myeloid leukemia tumor samples ispredictive of the patient response to cytotoxic chemotherapy (seeprofiling of AML). A set of normal tissues generally display lowvariance when profiled with the same technique (σ²<0.1). Some variancedetected in normal tissues could be reflective of subclinical pathology,as the technique would be expected to detect perturbations to individualcell signaling from immune responses and infections. These observationssuggested a working variance threshold for informative node states inarrayed flow cytometry experiments (σ²=0.1). This variance thresholdindicates that arrayed flow cytometry is extremely sensitive, andadvances in precision will further lower background noise and enablelower values for this threshold. In tumors, the variance for many nodestates is well above the threshold (median σ2 for significant nodes inprofiling of AML was 0.34). These results show that variance mapping oftumor signaling detects significant differences in the evolution oftumor signaling networks.

When clusters of signaling profiles have been identified it isfrequently useful to determine whether other factors, such as clinicalresponses, presence of gene mutations, and protein expression levels,are non-randomly distributed within the groups. If experiments orliterature suggest such a hypothesis in an arrayed flow cytometryexperiment, it can be judged with simple statistical tests, such as theStudent's t-test and the χ² test. Similarly, if two variable factorswithin the experiment are thought to be related, the r² correlationcoefficient from a linear regression is used to represent the degree ofthis relationship. Each of these tests is used to evaluate arrayed flowcytometry data as outlined below, and their appropriate use in analysisof a pool of tumor patient samples is introduced here.

Many hypotheses that are tested in arrayed flow cytometry experimentsask whether the values of a flow cytometry node state are significantlydifferent between two groups. To ask whether the means from twopopulations are significantly different, a Student's t-test is anappropriate way of obtaining a p-value. For example, as shown in theExamples below, we test the hypothesis that an increased response ofStat3 to G-CSF is found in patient samples with mutations in the Flt3gene. The Student's t-test is most frequently used in suchcross-platform comparisons of arrayed flow cytometry data with otherparameters. The t-value for such a test is calculated:

                      [Equation  1-3, t-value  for  two  means]${t\text{-}{value}} = {\frac{{\overset{\_}{x}}_{A} - {\overset{\_}{x}}_{B}}{\sqrt{\left( \frac{s_{A}}{N_{A}} \right) + \left( \frac{s_{B}}{N_{B}} \right)}}}$

In Equation 1-3, x _(A) is the mean of a node state across group A,s_(A) is the standard deviation of the node states across group A, andN_(A) is the number of samples in group A. The denominator of Equation1-2 is also referred to as the standard error. Together with the degreesof freedom (df), in this case is N−2, the t-value is used to look up thep-value for the hypothesis in a statistical table. In cases where thesamples are organized into discrete groups (e.g., “mutation positive”and “mutation negative”) for both parameters being compared it isappropriate to use a χ² test to determine a p-value. This test is mostcommon when comparing factors within signaling profile derived clustergroups, such as the presence or absence of gene mutation among sampleswith a specific signaling profile. A p-value calculated with a χ² testrepresents the degree to which a parameter was non-randomly distributedamong the groups. The χ² value for a set of parameters (p1, p2, p3, . .. ) for two groups (A and B) is calculated by comparing the observeddistribution to the expected distribution. The expected distribution forparameter p1 in group A (E_(A) ^(p1)) is calculated as:

              [Equation  1-4, expected  distribution  for  p 1  in  A]$E_{A}^{p\; 1} = {\left( \frac{O_{A}^{p\; 1} + O_{B}^{p\; 1}}{N} \right){\sum\; O_{A}}}$

In Equation 1-4, O_(A) ^(p1) is the observed number of p1 samples ingroup A, N is the total number of samples being compared, and ΣO_(A) isthe total number of samples observed in group A. The expected valuecalculated in Equation 1-2 is used with the observed value to calculatethe χ² value:

             [Equation  1-5, X²  test  for  an  observed  distribution]$X^{2} = {\sum\;\frac{\left( {O - E} \right)^{2}}{E}}$

The sum in Equation 1-5 is performed across all possible combinations ofparameters and groups. The resulting χ² value and the degrees of freedom(in this simple case, the number of parameters) are then used to look upthe p-value in a statistical table.

Generally, a p-value is considered significant when it is below athreshold value of α=0.05, indicating that the chance of the observedeffect taking place randomly was less than one in twenty. A p-value thatis equal to or less than 0.001 is generally taken to indicate theobservation is extremely significant. However, in some tests thethreshold value must be corrected. A potential danger of t-tests ariseswhen multiple t-tests are performed without first designating ahypothesis. For example, sometimes researchers split tumor patientgroups into groups (e.g., “tumor” and “normal” or “tumor A” and “tumorB”) and then test the significance of many parameters (e.g., expressionof 5,000 genes) to each of these groups. In this case, the probabilitythat the averages for any one gene would appear different due to chancehas increased with each gene being tested, and the α-value should bescaled accordingly. If the alpha value is not scaled, one may observefalse positives present among actual hits with higher p-values.

One straightforward way to scale an α-value is to divide it by thenumber of parameters or hypotheses being tested. Therefore, if 5,000factors were tested simultaneously within two groups, the significancethreshold should be set at the much more stringent value of α=0.00001.Other solutions to this issue employ groups of genes as hypothesisunits, lowering the number of tests, and the testing of pre-existinghypotheses. We adapted our profiling methods to avoid the perils ofmultiple t-tests by using variance mapping and unsupervised clustering(see AML profiling). Once groups are identified in this way, a χ² testcan be used to determine whether individual parameters (e.g.,chemotherapy response) were non-randomly distributed among the groups.In addition, following identification of signaling profiles usingvariance mapping, t-tests can be appropriately employed to determine thesignificance of individual hypotheses, such as the basal and potentiatedactivity of Stat5 in Flt3 wild type and mutant patient samples (see AMLprofiling).

A desired outcome in many arrayed flow cytometry experiments is thedetermination of how a network of signaling molecules function togetherto regulate cellular processes such as apoptosis and proliferation. Apowerful technique for visualizing multidimensional information employsself-organizing maps (SOMs) to sketch a picture for the researcher ofrelationships between parameters—in the present invention, signaltransduction node states. A basic algorithm to map relationship spaceamong parameters begins by determining the square of the Pearson productmomentum correlation coefficient (r) between the parameters in theanalysis:

                        [Equation  1-6, Pearson  coefficient]$r = \frac{N{\sum\;\left( {{XY} - {\left( {\sum X} \right)\left( {\sum Y} \right)}} \right)}}{\sqrt{\left( {\left\lbrack {{N{\sum X^{2}}} - \left( {\sum X} \right)^{2}} \right\rbrack\left\lbrack {{N{\sum Y^{2}}} - \left( {\sum Y} \right)^{2}} \right\rbrack} \right)}}$

In the application of Equation 1-5 to arrayed flow cytometry, X and Yare the fold MFI values from two node states for which the relationshipis being calculated, N is the number of samples, and the sum takes placefor all values in the set of samples. Those relationships that are abovea significance threshold (r²>0.5) are then used as a measure ofrelationship distance between the signaling nodes. Each related node isplaced, with some elasticity, at a distance of 1−r² from the nodes towhich it is related. This technique has been used to map therelationships between a set of leukemia nodes: Bcl-2 expression, p53accumulation, and five p53 phosphorylations (unpublished work ofJonathan Irish). This technique provides an intuitive map of signalingas a network of related nodes or node-states. SOMs are a powerful toolfor hypothesis generation and data visualization, but should not betaken to indicate causality. A continuing goal for arrayed flowcytometry SOMs is to represent clearly nodes and node states ondifferent levels of the analysis while still preserving therelationships between states.

Arrayed flow cytometry analysis of leukemia patient samples indicatesthat primary tissue samples differ strikingly from cells adapted to growin culture. In moving to a primary cell assay for signal transduction,we were surprised to find that primary tumor cells are not necessarilyconstitutively dysregulated, as was suggested by many years ofliterature on the subject (Benekli et al., 2002; Gouilleux-Gruart etal., 1997; Spiekermann et al., 2002). Instead, we understand now thattumor cells have developed signaling responses reflective of increasedproliferation, inhibition of apoptosis, and other tumor escapemechanisms. These pathways, which are revealed with environmental cues,are considered “potentiated.” Evaluating both basal and potentiatedstates of signal transduction nodes is a preferred method to elucidatetumor signaling pathology (see AML profiling).

With the development of response panels and a potentiated model ofsignaling we have found it useful to develop new language to describethe landscape of cell signaling. In addition, such pathologicalsignaling phenotypes can be used to classify human disease, stratifypatient risk, and, ultimately, to target disease therapies to patientgroups in whom they will be most effective. This generalized model ofpotentiated signaling outlines a central driving force behind theselective pressures that create tumors: gain proliferative signalswithout triggering cell death. In acute myeloid leukemia, suchproliferative signals take the form of potentiated responses of Stat5 tothe myeloid cytokines G-CSF, GM-CSF, and IL-3, and of Flt3 effectors toFlt3-ligand (see AML profiling), and a loss of apoptosis is achievedthrough Bcl-2 family member expression, loss of Stat1 signaling, changesin p38 MAPK regulation, and suppression of p53 activity (Jonathan Irish,unpublished data). The responses of these signaling nodes toenvironmental cues (e.g. potentiation) are very useful in theidentification of a tumor profile that was clinically relevant. Thus,the present invention shows that tumor cells that resist chemotherapycontain latent signals that are activated by environmental cues.Therefore, understanding dysregulated tumor signaling mechanisms caninclude evaluation of the expression levels of oncogenes and tumorssuppressors, as well as the capacity of signaling molecules to becomeactive.

There are several logical conclusions that can be derived from the threeclasses of node states outlined in the potentiated model. Potentiationof a node state among a group of cells or samples is relative to anexperimentally defined control group, such as a set of samples where thenode is non-potentiated or differently potentiated. The definition ofthe basal node state is relative to the experimental conditions andreflects the possibility that there may be unexpected factors affectingwhat is considered to be the basal state. Put another way, this model oftumor cell signaling requires an internal control for every node inevery sample's profile. As a practical result, a large portion of sampleto sample variability is removed from response panel profiles ofpotentiated signaling. Furthermore, under the potentiated model of tumorsignaling a set of experimental samples can be compared not only againsta putative normal set, but also against other tumors that displaydifferent signaling phenotypes. This is especially important when thenormal population is rare, was not obtained from a patient along withthe tumor, or when it is unclear what a normal precursor would be for aparticular tumor. As shown below (see AML profiling), this type ofpotentiated signaling model allows definition of tumor groups solely bydifferences in cell signaling.

Another deduction that can be made, based on the potentiated model, isif a node state in all or nearly all samples or cells reacts to a givenenvironmental cue in the same way, then it is not potentiated, despiteits ability to be responsive. While counterintuitive, it is an importantcomponent of the potentiated model. Practically, this means that todescribe a node state as potentiated, there must be a control populationof significant size within the cohort of profiled samples. And althoughthis population need not be defined a priori, it must be shown at somepoint to have significance to the experiment by identifying additionalparameters associated with the population (e.g., gene mutations, tumormarkers, clinical response).

This model of information nodes and the states that they occupy providesflexible terms to describe processes wherein cellular proteins adoptconformations, post-translational modifications, or localizations inorder to convey specific signals. In this context, a response panel usedto profile a diseased cell is being employed in the same way as a seriesof diagnostic commands a programmer uses to identify bugs in a faultycomputer program. In both cases, the inputs are chosen to maximize theinformation in the output and to reveal where instructions are beingincorrectly interpreted.

By identifying disrupted signaling nodes in tumors, accurate diagnosticsof therapeutic response and identification of molecular targets forpharmacological intervention can be developed. In many cases thedysregulated signal transduction of a tumor cell would signal forprogrammed cell death. These negative consequences of proliferativesignaling, which are normally carried out by apoptotic tumor suppressorpathways, are frequently lost or inactivated in tumors. In those tumorswhere apoptotic pathways are inactivated by another signal, such asexpression of anti-apoptosis molecules (e.g., Bcl-2, Bcl-XL, Toso),there is an opportunity to target the mechanism of anti-apoptosis andreactivate programmed cell death. Expression of some oncogenes,therefore, can be modeled as an accumulation of proliferative signalsthat send both pro-life and pro-death instructions simultaneously.Cooperating with these oncogenes are other mutations that disable thepro-death signaling consequences. This model is consistent with resultsindicating that reversal of oncogene expression leads to rapid death oftumor cells (Chin et al., 1999; Felsher, 2003).

Inherent to the potentiated model and arrayed flow cytometry methods isthe control of stimulated node states with the basal or resting state.This built in control enables a level of clarity that is difficult toobtain with other normalization techniques, where sample to samplevariations would dominate the profile (e.g., comparing each sample to anormal control). This is especially true for patient samples. It isimportant to note that there are frequently informative variations amongthe basal node states; the basal states are historically important totumor signaling and, in the example of acute myeloid leukemia, mostbasal states varied widely across samples (with the notable exception ofStat1, see AML profiling). In order to include the basal state of anode, one should place the basal values on the same scale as theresponses. This can be achieved by comparing each basal state to a fixedpoint among the tumor disease samples, such as the minimum or medianvalue observed

[Equation  1-7, scaled  basal  node  state]$x^{\prime} = {\log_{2}\left( \frac{x}{x_{\min}} \right)}$

Equation 1-7 will provide a spread of basal values on a comparable log₂scale, but should not be taken to indicate any information regardingchange from a normal state.

Hardware/General Techniques

It is understood by the skilled artisan that the steps of the assaysprovided herein can vary in order. It is also understood, however, thatwhile various options (of compounds, properties selected or order ofsteps) are provided herein, the options are also each providedindividually, and can each be individually segregated from the otheroptions provided herein. Moreover, steps that are obvious and known inthe art that will increase the sensitivity of the assay are intended tobe within the scope of this invention. For example, there may beadditionally washing steps, blocking steps, etc.

In a preferred embodiment, the reaction mixture or cells are containedin a well of a 96 well plate or other commercially available multiwellplate. In an alternate preferred embodiment, the reaction mixture orcells are in a FACS machine. Other multiwell plates useful in thepresent invention include, but are not limited to 384 well plates and1536 well plates. Still other vessels for containing the reactionmixture or cells and useful in the present invention will be apparent tothe skilled artisan.

The addition of the components of the assay for detecting the activationstate or activity of an activatable protein, or modulation of suchactivation state or activity, may be sequential or in a predeterminedorder or grouping under conditions appropriate for the activity that isassayed for. Such conditions are described here and known in the art.Moreover, further guidance is provided below (see, e.g., in theExamples).

In a preferred embodiment, the methods of the invention include the useof liquid handling components. The liquid handling systems can includerobotic systems comprising any number of components. In addition, any orall of the steps outlined herein may be automated; thus, for example,the systems may be completely or partially automated.

As will be appreciated by those in the art, there are a wide variety ofcomponents which can be used, including, but not limited to, one or morerobotic arms; plate handlers for the positioning of microplates;automated lid or cap handlers to remove and replace lids for wells onnon-cross contamination plates; tip assemblies for sample distributionwith disposable tips; washable tip assemblies for sample distribution;96 well loading blocks; cooled reagent racks; microtitler plate pipettepositions (optionally cooled); stacking towers for plates and tips; andcomputer systems.

Fully robotic or microfluidic systems include automated liquid-,particle-, cell- and organism-handling including high throughputpipetting to perform all steps of screening applications. This includesliquid, particle, cell, and organism manipulations such as aspiration,dispensing, mixing, diluting, washing, accurate volumetric transfers;retrieving, and discarding of pipet tips; and repetitive pipetting ofidentical volumes for multiple deliveries from a single sampleaspiration. These manipulations are cross-contamination-free liquid,particle, cell, and organism transfers. This instrument performsautomated replication of microplate samples to filters, membranes,and/or daughter plates, high-density transfers, full-plate serialdilutions, and high capacity operation.

In a preferred embodiment, chemically derivatized particles, plates,cartridges, tubes, magnetic particles, or other solid phase matrix withspecificity to the assay components are used. The binding surfaces ofmicroplates, tubes or any solid phase matrices include non-polarsurfaces, highly polar surfaces, modified dextran coating to promotecovalent binding, antibody coating, affinity media to bind fusionproteins or peptides, surface-fixed proteins such as recombinant proteinA or G, nucleotide resins or coatings, and other affinity matrix areuseful in this invention.

In a preferred embodiment, platforms for multi-well plates, multi-tubes,holders, cartridges, minitubes, deep-well plates, microfuge tubes,cryovials, square well plates, filters, chips, optic fibers, beads, andother solid-phase matrices or platform with various volumes areaccommodated on an upgradable modular platform for additional capacity.This modular platform includes a variable speed orbital shaker, andmulti-position work decks for source samples, sample and reagentdilution, assay plates, sample and reagent reservoirs, pipette tips, andan active wash station.

In a preferred embodiment, thermocycler and thermoregulating systems areused for stabilizing the temperature of heat exchangers such ascontrolled blocks or platforms to provide accurate temperature controlof incubating samples from 0° C. to 100° C.

In a preferred embodiment, interchangeable pipet heads (single ormulti-channel) with single or multiple magnetic probes, affinity probes,or pipetters robotically manipulate the liquid, particles, cells, andorganisms. Multi-well or multi-tube magnetic separators or platformsmanipulate liquid, particles, cells, and organisms in single or multiplesample formats.

In some embodiments, the instrumentation will include a detector, whichcan be a wide variety of different detectors, depending on the labelsand assay. In a preferred embodiment, useful detectors include amicroscope(s) with multiple channels of fluorescence; plate readers toprovide fluorescent, ultraviolet and visible spectrophotometricdetection with single and dual wavelength endpoint and kineticscapability, fluoroescence resonance energy transfer (FRET),luminescence, quenching, two-photon excitation, and intensityredistribution; CCD cameras to capture and transform data and imagesinto quantifiable formats; and a computer workstation.

In a preferred embodiment, the detecting is by FACS. In another aspect,the detecting is by high-pressure liquid chromatography (HPLC), forexample, reverse phase HPLC, and in a further aspect, the detecting isby mass spectrometry.

These instruments can fit in a sterile laminar flow or fume hood, or areenclosed, self-contained systems, for cell culture growth andtransformation in multi-well plates or tubes and for hazardousoperations. The living cells may be grown under controlled growthconditions, with controls for temperature, humidity, and gas for timeseries of the live cell assays. Automated transformation of cells andautomated colony pickers may facilitate rapid screening of desiredcells.

Flow cytometry or capillary electrophoresis formats can be used forindividual capture of magnetic and other beads, particles, cells, andorganisms.

The flexible hardware and software allow instrument adaptability formultiple applications. The software program modules allow creation,modification, and running of methods. The system diagnostic modulesallow instrument alignment, correct connections, and motor operations.The customized tools, labware, and liquid, particle, cell and organismtransfer patterns allow different applications to be performed. Thedatabase allows method and parameter storage. Robotic and computerinterfaces allow communication between instruments.

In a preferred embodiment, the robotic apparatus includes a centralprocessing unit which communicates with a memory and a set ofinput/output devices (e.g., keyboard, mouse, monitor, printer, etc.)through a bus. Again, as outlined below, this may be in addition to orin place of the CPU for the multiplexing devices of the invention. Thegeneral interaction between a central processing unit, a memory,input/output devices, and a bus is known in the art. Thus, a variety ofdifferent procedures, depending on the experiments to be run, are storedin the CPU memory.

These robotic fluid handling systems can utilize any number of differentreagents, including buffers, reagents, samples, washes, assay componentssuch as label probes, etc.

The following example serves to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are expressly incorporated by reference intheir entirety, including patent application U.S. Ser. No. 10/193,462,filed July 2002, now U.S. Pat. 7,563,584, which claims the benefit ofthe filing dates of U.S. Ser. Nos. 60/304,434 and 60/310,141.

EXAMPLES Example 1

In this Example, using the methods and compositions of the presentinvention, the present inventors (also referred to herein as “we”) showthat Leukocyte Function Antigen-1 (LFA-1) is essential in the formationof immune cell synapses and has a role in the pathophysiology of variousautoimmune diseases. In this Example, using the methods and compositionsof the present invention, the present inventors demonstrate that ICAM-2induced an LFA-1 signal transduction pathway that is linked to receptorclustering and activation by both the microtubule and actincytoskeleton. ICAM-2 exhibited a 21.7 pM/cell binding affinity asdetermined by single cell analysis. ICAM-2/LFA-1 engagement inducedactivation of PKC and a reorganization of both the actin and microtubulecytoskeleton. These events resulted in a Syk dependent activation of thep44/42 MAPK pathway upon cytotoxic T cell effector-target cell bindingvia active LFA-1. ICAM-2 mediated human CD56⁺CD8⁺ perforin release andresultant cytotoxicity to target leukemia cells. In comparison to theother ICAMs, ICAM-3 was found to be most similar to ICAM-2's effect anddissimilar to ICAM-1. In IL-2 pre-activated human PBMC,ICAM-2>ICAM-3>>ICAM-1 in mediating perforin release of a CD56⁺CD8^(med)population. All ICAMs contributed to perforin and granzyme-A loss inCD56⁺CD8^(high) populations. These results identify a specificfunctional consequence for ICAM-2/LFA-1 in subset-specific cytotoxic Tcell immunity.

Introduction

Leukocyte Function Antigen-1 (LFA-1) is an α,β heterodimer integrininvolved in leukocyte adhesion (van Kooyk, Y., and Figdor, C. G. (2000)Curr Opin Cell Biol 12, 542-547). At present, it is well understood thatLFA-1 participates in lymphocyte adhesion, with prominent roles in theformation of the immunological synapse (Dustin, M. L., and Shaw, A. S.(1999) Science 283, 649-650), and lymphocyte extravasation andrecirculation (Volkov, Y., et al., (2001). Nat Immunol 2, 508-514).LFA-1 adhesion is governed by the intercellular adhesion molecule(ICAMs)-1, -2, and -3 ligands (van Kooyk and Figdor, 2000). Patientsafflicted with Leukocyte Adhesion Deficiency disorder (LAD), a syndromein which the LFA-1 integrin is mutated or missing, suffer severrecurrent bacterial infections and impaired overall immunity (Bunting,M., et al., (2002) Curr Opin Hematol 9, 30-35). Among these clinicalmanifestations, the LFA-1 knockout mouse has suggested that LFA-1 mayhave a potential role in mediating tumor regression in adoptiveimmunotherapy (Mukai, S., et al., (1999) Cell Immunol 192, 122-132;Nishimura, T., et al., (1999) J Exp Med 190, 617-627). Although thesestudies genetically link a lymphocyte adhesion molecule with impairedimmune function, the molecular details that mediate theseimmunopathologies are less well understood.

Investigations of LFA-1 have primarily focused on the integrin'sadhesive role. It is unclear as to how the physical processes of LFA-1integrin activation and receptor clustering are interconnected andtranslated into cellular signals upon ligand binding. It is lessunderstood how the absence of these events leads to the devastatingeffects of LAD and the impaired immune responses in LFA-1 knockout mice.We therefore sought to decipher the molecular details of a modelinteraction of ICAM-LFA-1 to understand LFA-1 signaling mechanismsinitiated upon cell-to-cell contact. Utilizing multiparameter singlecell analysis to monitor LFA-1 receptor dynamics upon treatment with asoluble ICAM-2, we found that both the actin and the microtubulecytoskeleton couple ICAM-2 adhesion to LFA-1 activation and clustering.The microtubule cytoskeleton constrained the LFA-1 conformational change(activation), an event that preceded LFA-1 clustering as measured bymultiparameter flow cytometry. The induced LFA-1 activation led to theactivation of the p44/42 Mitogen Activated Protein Kinase pathway (MAPK;RAF/MEK/ERK), an event that was dependent on both Pyk2 and Syk kinaseactivities.

The present inventors investigated these molecular details of the ICAM-2mediated LFA-1 activation in the adhesion between cytotoxic T cells anda target leukemia cell, an event that requires cell-to-cell contact.ICAM-2 stimulation of human CD56⁺CD8⁺ T cells could induceperforin/granzyme-A mediated cytotoxicity of leukemia cells. Thisdirected killing was shared by ICAM-3 and to a lesser extent by ICAM-1,two other LFA-1 ligands. These results distinguish a signaling mechanismfor ICAM-2/LFA-1 directed cytotoxic T lymphocyte immunity and suggestpossible mechanisms by which tumor secretion of ICAM-2 and possiblyICAM-3 might allow for evasion of a directed cytotoxic T cell immuneresponse.

Results

Recombinant ICAM-2 Promotes LFA-1 Mediated Adhesion

The present inventors chose a model Jurkat T cell line as a system toinitially dissect the LFA-1 signaling mechanism and then verified thefindings in human T cells. A biochemically purified ICAM-2 protein wasproduced to study ICAM-2/LFA-1 interactions in the absence of otherligands. We purified human ICAM-2 from retrovirally transduced NIH3T3cells using immunoaffinity chromatography and subsequent gel filtration.We compared it to an ICAM-2-FC fusion protein produced in NSO murinemyeloma cells. These murine-based mammalian expression systems werechosen on the basis that they yielded a bioactive form of ICAM-2.Biochemical analysis of ICAM-2FC protein was consistent with theexpected molecular weight of the fusion protein (76 kD) and purifiedhuman ICAM-2 displayed a molecular weight of 72-74 kD. This size wassimilar to the 75 kD ICAM-2 purified from Jurkat T cells (data notshown).

The present inventors generated a FITC conjugated ICAM-2 (ICAM-2-FITC)to study LFA-1 receptor dynamics by flow cytometry and laser scanningconfocal microscopy (LSCM). We tested for ligand binding of the LFA-1receptor by monitoring the binding kinetics of ICAM2-FITC on singlecells. Low binding was observed in the first 150 seconds, whereuponthere was a progressive increase until 750 seconds, and leveledthereafter. In contrast, an anti-LFA-1 antibody displayed an initialspike in the first 50-100 seconds and equilibrated until 800 seconds.Pre-activating LFA-1 by treatment with PMA (McDowall, A., et al., (1998)J Biol Chem 273, 27396-27403) showed an immediate binding of ICAM-2. Thegradual ICAM-2 binding after 150 seconds suggested an enhanced LFA-1binding for its ICAM-2 ligand after some binding-induced event—aproperty not observed using the anti-LFA-1 or upon PMA activated LFA-1.Binding of ICAM-2-FITC was not observed in trypsinized cells (data notshown) and was blocked by antibodies to LFA-1 (described below).Therefore, there appeared to be an increase in binding of the ICAM-2ligand as a function of time, suggesting the presence of an inducedbinding site on the target cells.

Analysis of the ICAM-2 binding population by flow cytometry showed adependency on both the actin cytoskeleton and temperature. ICAM-2adhesion was enhanced at 37° C. vs. 4° C. Pre-treatment with the actindepolymerizing agent cytochalisin D revealed two ICAM-2 bindingpopulations at both 37° C. and 4° C., contrasting with the bindingphenomena observed for anti-LFA-1. Saturation of ICAM-2-FITC wasobserved at 37° C. more readily than at 4° C. (data not shown). Singlecell binding affinity measurements for ICAM-2 were obtained by computingthe percent ICAM-2-FITC bound per cell. Curve fit analysis indicated adissociation constant of 0.21±0.07 M/10⁴ cells. This value equates to21.7 pM/cell, representing the first ligand binding measurementsreported for ICAM-2 within the physiological context of cell surfaceLFA-1. Thus, quantitative single cell analysis of ICAM-2 ligand bindingsuggests strong binding at physiological temperatures.

Soluble ICAM-2 Induces LFA-1 Clustering and Cytoskeleton Polarization

The present inventors investigated if LFA engagement alteredcytoskeletal structures and observed a reorganization of both the actinand microtubule cytoskeleton upon ICAM-2 stimulus. The present inventorsmonitored the cytoskeletal architecture by flow cytometry and observed asimultaneous change in the actin and microtubule organization uponICAM-2 binding, an effect consistent with depolymerization. ICAM-2treatment induced a rapid clustering of LFA-1 within one minute, withmultiple clustering events at five minutes. Using the ICAM-2-FITC ligandto visualize the cell surface, indicated that the ICAM-2 ligand inducedclustering of the LFA-1 receptor. The clustering event showed somecolocalization using a non-blocking 2 integrin antibody (clone CTB104).Thus, we speculated that ICAM-2 binding to LFA-1 induced a signal thatresulted in a reorganization of the LFA-1/ICAM-2 complex. We thereforedecided to investigate this in relation to the observed changes in theactin/microtubule cytoskeleton.

The present inventors assessed LFA-1 receptor dynamics by multiparameterflow cytometry upon ICAM-2 binding to correlate LFA-1 activation andclustering. We utilized the doublet discriminator module on aFACSCalibur machine to distinguish between distributed and focalizedfluorescence pulses (FFP) upon laser excitation of single cells.Incubation of ICAM-2 at 37° C. vs. 4° C. displayed a decrease in theFFP, an effect that was greatly enhanced upon cytochalisin D treatment.ICAM-2-FITC surface binding was monitored by the fluorescence intensityand normalized against the time-of-flight (TOF) of the fluorescencepulse (FP). We interpreted the value of ICAM-2-FITC intensity per TOF asa quantitative assessment for LFA-1 clustering, as the TOF isproportional to the laser-excited cellular area. Computing this value asa function of time for an ICAM-2 stimulus is proportional to theincreased clustering events observed by LSCM.

ICAM-2 Adhesion Induces a Conformational Change in LFA-1 that isRegulated by the Microtubule Cytoskeleton

Although the enhanced ICAM-2 adhesion and induced LFA-1 clustering isreflective of overall increased avidity for the ICAM-2 ligand, it doesnot necessarily reflect an LFA-1 activation state (high affinity state)(McDowall et al., 1998). Upon LFA-1 activation, a conformational changeexposes an epitope that is recognized by the mAb24 antibody (Neeson, P.J., et al., (2000) J Leukoc Biol 67, 847-855). A mAb24-Alexa633conjugate was used to assess the activation state of LFA-1 upon ICAM-2stimulus by flow cytometry. Unstimulated cells did not display mAb24binding, contrasting the induction observed with PMA treatment. ICAM-2stimulated cells displayed a bimodal population in active LFA-1, aneffect that was attenuated by cytochalisin D. Treatment with microtubuledisrupting agents, nocodazole and taxol, resulted in full activation ofLFA-1 upon ICAM-2 stimulus. In contrast, disrupting the actincytoskeleton via cytochalisin D diminished the ICAM-2 induced LFA-1activation, although it enhanced LFA-1 receptor clustering andsubsequent ICAM-2 binding. Therefore, the actin and microtubulecytoskeletal network differentially impact LFA-1 activity and avidity.

The present inventors monitored LFA-1 activation and clusteringsimultaneously as a function of ICAM-2 stimulus per time by flowcytometry. Correlating the mean fluorescence of mAb24 antibody with theLFA-1 clustering value revealed LFA-1 activation preceeded LFA-1clustering within 30 seconds there was a significant increase in bindingof the mAb24 antibody but only a modest increase in clustering. However,after another 30 seconds up to 30 minutes the relative binding of mAb24increased somewhat but there was a significant increase in theclustering value. Thus, these results suggest that the ICAM-2 ligandinduced activation of LFA-1 is followed by subsequent LFA-1 clustering.

The present inventors observed that treating cells with a PKC inhibitor,bisindolymaleimide I (BIM I), inhibited ICAM-2 induced LFA-1 activationas measured by using mAb24 binding. ICAM-2 adhesion, as measured by thebinding of ICAM-2-FITC, was not affected. This suggested that the ligandinduced receptor conformational change was dependent on intracellularkinases. Interestingly, ICAM-2 induced a calcium influx, a componentnecessary in PKC activation (data not shown). Thus, these observationssuggest that the ICAM-2 ligand induced exposure of the mAb24 neoepitopetriggers a PKC dependent intracellular signaling event. We decided toinvestigate the downstream signaling consequences of ICAM-2 binding toLFA-1.

ICAM-2 Induces p44/42 MAPK Activity Through LFA-1

Flow cytometric based kinase profiling experiments were performed toidentify a signaling pathway downstream of PKC activation upon ICAM-2stimulus. Treatment with ICAM-2 induced both p44/42 MAPK phosphorylationand activation. An ICAM-2 titration correlated with phosphorylation ofp44/42 MAPK as determined by single cell flow cytometric analysis,results congruent with kinase activity analysis. Titration of mAbs tospecific integrins competed with ICAM-2 binding, and thus diminished theinduced p44/42 MAPK phosphorylation. This inhibition was not observedafter pretreatment with mAbs to various integrins indicating that theICAM-2/LFA-1 interaction was mediating the p44/42 MAPK activation.

Activation of PKC, PYK2, and SYK are Necessary for the ICAM-2/LFA-1Induction of p44/42 MAPK Activity

The present inventors undertook flow cytometric based p44/42 MAPK kinaseinhibition and activation profiling to identify necessary components forLFA-1 signaling. PKC inhibitor BIM I, cytoskeletal disrupting agentscytochalisin D, taxol, nocodozole, and sequestering of divalent cationsby EDTA diminished the ICAM-2 induced p44/42 MAPK signal, suggestingthat the ligand-induced events of LFA-1 are mechanically linked tosignal transduction by the actin-microtubule cytoskeleton. To identifyupstream kinases that were responsible for signal transmission fromLFA-1 to p44/42 MAPK, a series of kinase inhibitors were applied andtested for their ability to abrogate the ICAM-2 induced p44/42 MAPKactivity, whereas Herbimycin A and Emodin, inhibitors of src and p56lckhad no effect. Tyrphostin A9 and piceatannol, specific inhibitors ofproline-tyrosine kinase 2 (Pyk2) and Spleen-tyrosine kinase (Syk),respectively (Avdi, et al., (2001) J Biol Chem 276, 2189-2199; Fuortes,et al., (1999) J Clin Invest 104, 327-335) abrogated the ICAM-2 inducedactivation of p44/42 MAPK and its upstream activator Raf-1.

The present inventors tested whether Pyk2 and Syk interacted with aparticular integrin. Pyk2 and Syk were phosphorylated andco-immunoprecipitated with the integrin upon ICAM-2 treatment,indicating Pyk2 and Syk translocated to the membrane. This wascoincident with phosphorylation of Pyk2 and Syk upon ICAM-2 stimulus asa function of time. Phosphorylation of PKC/_(II), and Pyk2 were detectedat one minute, followed by Syk phosphorylation at 5 minutes. Weconfirmed that Pyk2 and Syk activities were dependent on PKC activation.Taken together with the above results, this suggested that the LFA-1signaling mechanism imparted by ICAM-2 is at least initiated by PKC andrelayed to the p44/42 MAPK pathway by Pyk2 and Syk.

LFA-1 is Involved in Effector-Target Cell Adhesion and Facilitates HumanCytotoxic T Cell Activation

Since LFA-1 is involved in adhesion between lymphocytes, a process thatoccurs at several immunological synapses, we were interested ininvestigating the molecular events identified for the ICAM-2/LFA-1interaction in a physiological context. It has been suggested that aclustered topographic presentation of ICAM-2, independent of expressionlevels, is an effective target structure by which natural killer cellsinitiated cytotoxicity (Helander, T. S., et al., (1996) Nature 382,265-268). We first applied a FACS based effector-target killing assay toquantitatively monitor target cell lysis of HL60 leukemic cells upontreatment with stimulated human PBMC at various effector: target cellratios. Flow cytometric detection of target cell lysis has been reportedto be more sensitive than the standard chromium release assays (Lecoeur,H., et al., (2001) J Immunol Methods 253, 177-187). We labeled HL60cells with the fluorescent dye CFSE and monitored the cell quantity byflow cytometry in standard effector-target cell based assays. SolubleICAM-2 could initiate target cell lysis in the presence of IL-2 but notin the absence of IL-2. In IL-2 pre-activated cells, ICAM-1 and ICAM-3did not initiate as potent a cytotoxic cell response in contrast toICAM-2.

Since natural killer cells (NK) comprise a heterogeneous population,namely specific cytotoxic T lymphocytes (CTL, with C8⁺ subsets therein),NK cells (CD16⁺ and subsets therein), and CD4⁺ TH1 cells (Biron, C. A.,and Brossay, L. (2001) Curr Opin Immunol 13, 458-464), we determined ifICAM-2 was unique to a particular human NK cell subset. We utilized themultidimensional gating capability of flow cytometry to identifydistinct cellular populations that were contributing to the cytolyticactivity observed in human PBMC. We also monitored intracellular levelsof perforin and granzyme-A by flow cytometry, two proteins that mediatetarget cell lysis by NK cells in these populations. We identified 6distinct populations by CD8 and CD56 surface stains in human PBMC andgated on these subsets for all subsequent intracellular functionalassays. We performed effector-target cytotoxicity assays in the presenceof ICAM-1, ICAM-2, and ICAM-3 soluble ligand and HL60 target cells. Wedid not observe significant changes in population subset frequenciespost stimulation. The CD56⁺CD8^(low) population displayed no significantchanges in intracellular perforin or granzyme-A upon stimulation withICAM-1, -2, or -3. The CD56⁺CD8^(med) population displayed a slightincrease (1.5-2 fold) in the frequency of the perforin negativepopulation for ICAM-2 and ICAM-3 (21.5% ICAM-2>19.8% ICAM-3>13.7%ICAM-1). The CD56⁺CD8^(high) population displayed a loss in bothgranzyme-A and perforin for ICAM-1, -2, -3 stimulations compared tounstimulated with a significant loss in the granzyme-A negativepopulation for ICAM-2 (58.3%) compared to ICAM-1 (4.12%) or ICAM-3(3.07%). The CD56⁻CD8^(high) also displayed a loss of both granzyme-Aand perforin by all ICAM stimulations. Since it was not possible topositively identify the subsets within the CD56⁻CD8⁻ population, theywere omitted from analysis.

Quantifying the intracellular amounts of perforin and granzyme-A in theCD56CD8 subsets relative to unstimulated cells also identifiedsimilarities and differences for the ICAMs as evidenced below. ICAM-2and ICAM-3 mediated loss of granzyme-A and perforin to a greater extentthan ICAM-1. Additionally, in IL-2 pre-activated cells, differenceswhere seen with the ICAM stimulations: ICAM-2>ICAM-3>>ICAM-1 displayed aloss of perforin, particularly in the CD56⁺CD8^(med/high) populations.ICAM-2 and ICAM-3 also induced perforin loss in the CD56⁺CD8^(low),however ICAM-2 required preactivation by IL-2. There were lower levelsof granzyme-A detected for the CD8^(high) subsets (CD56⁺ or CD56⁻) forICAM-2>ICAM-3>ICAM-1>unstimulated. In the presence of IL-2pre-activation, all the ICAMs induced release of granzyme-A in theCD56⁺CD8^(high/med) populations, with a particular decrease by ICAM-2.No significant changes were seen in the CD56⁺CD8^(low) population forgranzyme-A. These differences were similar at various effector-targetcell ratios (50:1, 25:1, 12.5:1) (data not shown). Thus, similaritiesand difference exist for ICAM-1, -2, and -3 stimulation of cytolyticactivity in CD56CD8 subsets. All three ICAMs mediated perforin releasein the CD56⁺CD8^(high) populations. ICAM-2 and ICAM-3 were most similarin mediating perforin/granzyme-A release in the CD56⁺CD8^(high) andCD56⁺CD8^(med) populations.

We focused on the CD56⁺CD8⁺ cells (both the CD8^(med) and CD8^(high)subsets) and tested if inhibition of Syk, p44/42 MAPK or disruption ofthe cytoskeleton detrimentally affected effector-target (E:T) cellconjugation as measured by a flow cytometric conjugate formation assay(Morgan, M. M., et al., (2001) J Immunol 167, 5708-5718). Disruption ofcytoskeletal actin and microtubules enhanced E:T conjugate formationcongruent with prior results that disruption by these agents enhancedLFA-1 activation. Inhibition of Syk by piceatannol inhibited conjugateformation whereas inhibiting p44/42 MAPK by PD98059 did not. Theseresults suggest that Syk activity is necessary for LFA-1 adhesion ofeffector-target cells and is consistent with a report indicating thatSyk/ZAP-70 are necessary for LFA-1 to LFA-1 activation on the same cell(Soede, R. D., et al., (1999) J Immunol 163, 4253-4261). p44/42 MAPKappeared to not be necessary for E:T conjugate formation. Monitoringactive LFA-1 and intracellular activation of p44/42 depicted a timedependent correlation between these two markers in CD56⁺CD8⁺ cells asstimulated by ICAM-2.

Discussion

In this report it was observed that (1) ICAM-2 can induce LFA-1clustering, activation, and cytoskeletal reorganization in the absenceof exogenous activators such as cytokines or TCR signaling; (2) LFA-1transmits a signal to the p44/42 MAPK pathway involving PKC, Pyk2, andSyk upon ligand binding; and (3) LFA-1 receptor dynamics aremechanically coupled to signal transduction by both the actin andmicrotubule cytoskeleton network. The physiological outcome of thesemolecular events triggered perforin and granzyme A mediated CD56⁺CD8⁺ Tcell cytotoxicity that were mostly shared by ICAM-2 and ICAM-3 but notICAM-1.

2 integrin signaling mechanisms vary depending on the system of studyand are centered on adhesive roles in cell morphology and motility (Dib,K. (2000) Front Biosci 5, D438-451). 2 integrin signaling has been shownto involve cytoskeletal reorganization via tyrosine phosphorylation ofpaxillin, vav, and GTPase activating proteins among others (Fuortes, M.,et al., (1994) J Cell Biol 127, 1477-1483; Zheng, L., et al., (1996)Proc Natl Acad Sci USA 93, 8431-8436). Studies focused on LFA-1 mediatedleukocyte adhesion (CD11a/CD18) have shown a regulatory role for PKC inLFA-1 avidity (Bleijs, D. A., et al., (2001) J Biol Chem 276,10338-10346; Hedman, H., and Lundgren, E. (1992) J Immunol 149,2295-2299) and have demonstrated that TCR signaling can activate LFA-1(Peterson, E. J., et al., (2001) Science 293, 2263-2265). It has alsobeen shown that chemokines, in the absence of TCR signaling, can serveas activators of LFA-1 during lymphocyte/endothelial contact(Constantin, G., et al., (2000) Immunity 13, 759-769). It has not beenclear how LFA-1 integrin adhesion, clustering, and activation arecoupled to intracellular signaling events, in the absence of external(chemokine) or internal (TCR or costimulatory molecule) stimulation.

A synthesized peptide of ICAM-2's first Ig domain (P1, amino acids21-42) can induce LFA-1 mediated adhesion at high concentrations (62 M),which was comparable to a 48-fold lower ICAM-2 soluble proteinconcentration (1.3 M) in a bulk cellular adhesion assay (Kotovuori, A.,et al., (1999) J Immunol 162, 6613-6620). However, P1 binding did notinduce the active conformation of LFA-1 and did not induce calciuminflux (Kotovuori et al., 1999), whereas full length ICAM-2 bindingresulted in active LFA-1 (see FIG. 6D from USSN 10/193,462) and acalcium influx event (data not shown). The calculated ICAM-2 affinity of217±66 nM (per 10⁴ cells) contrasts the 605±55 nM k_(D) reported usingBIAcore analysis of an engineered “active” locked I domain of LFA-1(Shimaoka, M., et al., (2001) Proc Natl Acad Sci USA 98, 6009-6014). Thereported affinities for ICAM-2 binding here take advantage of singlecell resolution within a physiological context, something not possibleutilizing purified or genetically engineered LFA-1. The differencesobserved for peptide vs. protein concentrations are likely attributed toimpurities in the peptide synthesis and/or presence of carbohydratemoieties native to the endogenous ICAM-2, which comprise greater than 30kD of its approximate 66 kD molecular weight and have been suggested toorient ICAM-2 binding to LFA-1 (Casasnovas, J. M, et al. (1997) Nature387, 312-315; de Fougerolles, A. R., et al. (1991) J Exp Med 174,253-267).

We investigated the role of the actin and microtubule cytoskeleton inLFA-1 receptor activation and clustering as induced by the ICAM-2 ligandby multiparameter flow cytometry. Disruption of the actin cytoskeletonenhanced LFA-1 clustering and ICAM-2 binding, corroborating previousstudies that suggested the actin cytoskeleton constrains LFA-1 mobility(Lub, M., et al., (1997) Mol Biol Cell 8, 341-351). Interestingly, actindepolymerization abrogated the ICAM-2 induced LFA-1 activation. Incontrast, disruption of the microtubules by both nocodazole and taxolenhanced LFA-1 activation as determined by exposure of the neo-epitoperecognized by the mAb24. Recently, it has been reported thatdepolymerization of microtubules increases the lateral mobility of 2integrins in macrophage cell lines (Zhou, X., et al., (2001) J Biol Chem276, 44762-44769); therefore its conceivable that the microtubulesregulate the conformational change upon ligand binding necessary forexposure of the LFA-1 activation epitope. These observations suggest theactin-microtubule cytoskeleton regulates both the high-avidity and highaffinity state of LFA-1 upon ligand binding. We observed that LFA-1signal transduction was abrogated in the presence of all cytoskeletaldisrupting agents tested (cytochalisin D, nocodazole, and taxol)indicating that the LFA-1 receptor is linked to signal transductionmachinery by the cytoskeleton. Thus, the mechanistic uncoupling of thehigh avidity and high affinity states of LFA-1 suggests thatintracellular events that regulate/mediate these two states exist at theLFA-1 integrin-cytoskeletal juncture and relay the LFA-1 receptordynamics to intracellular signaling proteins upon ligand binding.

Several chemical inhibition screens were designed to identify theproteins involved in the LFA-1 to p44/42 MAPK signaling event. Both Pyk2and Syk were identified to be necessary for activation of the p44/42MAPK pathway and were dependent on PKC activity upon ICAM-2 binding.Phosphorylation of Pyk2 has been associated with homotypic adhesionmediated by an LFA-1/ICAM-1 interaction in B cells (McDonald, J. T., etal., (2000) Immunol Invest 29, 71-80). In addition, Pyk2 activation hasbeen shown to be necessary for p44/42 MAPK activity in other modelsystems (Barsacchi, R., et al., J. (1999) FEBS Lett 461, 273-276; Lev,S., et al., (1995) Nature 376, 737-745). Syk is a tyrosine kinaseessential in III 3 signaling (Saci, A., et al., (2000) Biochem J 351 Pt3, 669-676), and links Fc R1 signaling to the ras/MAPK pathway(Jabril-Cuenod, et al., (1996) J Biol Chem 271, 16268-16272). Inhibitionor ablation of Syk, either by pharmacological means (via inhibition bypiceatannol), biochemical means (dominant negative Syk), or geneticmeans (Syk^(−/−) mice) inhibits natural cytotoxicity (Brumbaugh, K. M.,et al., (1997) J Exp Med 186, 1965-1974; Colucci, F., et al., (1999) JImmunol 163, 1769-1774). Thus LFA-1 activation signaling to Syk, akinase that has been shown to be important for NK cell function,provides a biochemical link between surface integrin activation andeffector cell function.

The present inventors demonstrated that both Pyk2 and Syk are necessaryin ICAM-2 induced LFA-1 signaling to Raf-1, the upstream kinase in thep44/42 MAPK (RAF/MEK/ERK) cascade. Inhibition of p44/42 MAPK did notprevent the occurrence of CD56⁺CD8⁺ cell conjugation. Byimmunofluorescence analysis, it has been shown that treatment of the NKleukemic cell line YT with the p44/42 MAPK inhibitor PD98059 inhibitsperforin redistribution to the site of effector-target cell contact(Wei, S., et al., (1998) J Exp Med 187, 1753-1765). In addition, thep44/42 MAPK pathway has been shown to be important in the regulation ofcytoxicity in natural killer cells (Jiang, K., et al., (2000) NatImmunol 1, 419-425). Thus, the p44/42 MAPK pathway, here demonstrated tobecome active upon LFA-1/ICAM-2 binding, has been shown to be connectedto at least perforin granule exocytosis. Thus, the LFA-1 signalingpathway as elicited by ICAM-2 contains signaling junctures that map toboth the effector-target cell adhesion event and activation of cytolyticmachinery in the human CD56⁺CD8⁺ cytotoxic T cell population. Theseresults provide direct evidence for a functional consequence of LFA-1integrin adhesion with cytolytic signaling mechanisms.

We also observed that ICAM-2 was similar to ICAM-3 in mediatingcytolytic activity as evidenced by release of perforin and granzyme-A ineffector-cell conjugation, effects of which contrasted ICAM-1 (see FIG.8 from USSN 10/193,462). We have previously observed similaritiesbetween ICAM-2 and ICAM-3 intracellular signaling mechanism that alsodiffered from that of ICAM-1 (Perez, O.D., et al., (2002) Immunity 16,51-65). However, the results do not exclude the possibility of ICAM-2stimulating other yet to be identified cytotoxic capable subsets, ashigh cytolytic activity was observed in bulk PBMC (see FIG. 9 from USSN10/193,462).

Prior investigations into cytotoxic T cells have established thatblocking the LFA-1/ICAM interactions inhibits effector-target celladhesion and therefore concluded that it also blocks cytolytic activityin NK cells (Donskov, F., et al., (1996) Nat Immun 15, 134-146; Krensky,A. M., et al., (1984) J Immunol 132, 2180-2182; Matsumoto, G., et al.,(2000) Eur J Immunol 30, 3723-3731). Functional studies of NK cells fromLFA-1^(−/−) mice have demonstrated that LFA-1 adhesion is necessary forIL-2 activated NK killing (Matsumoto et al., 2000) and also thatLFA-1^(−/−) CD8⁺ T cells are defective for T cell activation andeffector function (Shier, P., et al., (1999) J Immunol 163, 4826-4832).Interestingly, NK cell cytotoxicity is defective in NK cells from LADpatients (Shibuya, K., et al., (1999) Immunity 11, 615-623). It has onlyrecently been shown that the directed killing of cytotoxic T lymphocytesinvolves polarization of the microtubule-organizing center (MTOC)towards LFA-1 at the CTL-target site (Kuhn, J. R., and Poenie, M. (2002)Immunity 16, 111-121), an indication that LFA-1 may possess a functionalrole other than strictly adhesion.

In conclusion we find that ICAM-2, as an LFA-1 ligand, can mediateactivation and clustering of the LFA-1 receptor—an event that in turnpolarizes the microtubule and actin cytoskeleton and activates thep44/42 MAPK pathway. These events were found to be necessary foreffector-target cell binding of CD56⁺CD8⁺ T cells, and perforin/granzymeA mediated cytolytic activity. This effect was shared by ICAM-3. Themechanisms governing LFA-1 receptor dynamics and intracellular signalingreported here suggest LFA-1 signaling functionally contributes inCD56⁺CD8⁺ cytolytic activity in addition to possessing an adhesive roleupon which other molecular interactions occur. Improper localization ofthe MTOC has been shown to inhibit exocytosis of lytic granules in CD8⁺tumor infiltrating T cells, thereby ablating perforin mediated cytolyticactivity necessary for a CTL response in murine tumor models (Radoja,S., et al., (2001) J Immunol 167, 5042-5051). Ironically, defective CD8⁺tumor infiltrating T cells can effectively mediate cell killing in vitro(Radoja, S., et al., (2001) J Immunol 166, 6074-6083), suggesting tumormediated inhibitory mechanisms exist within the tumor microenvironment.The production of soluble ICAMs (1 and 3) has been observed in sera fromcancer and autoimmunity patients, though analysis has not been extendedto ICAM-2 (Bloom, et al., (2002) J Rheumatol 29, 832-836). Only onereport has indicated that elevated levels of soluble ICAM-2 were presentin leukemia patients and decreased upon chemotherapy (Mustjoki, S., etal., (2001) Br J Haematol 113, 989-1000). The etiology of theseobservations is unknown. In the context of the work presented here, itis plausible to speculate that either dysregulation of surface ICAM-2 orsecretion of soluble ICAM-2 can prematurely trigger or block CD56⁺CD8⁺cytolytic activity at the effector-target site and permit tumor escapefrom T cell lysis. Other, specific roles, of ICAM-2 in its interactionwith other integrin ligands could lead to a better understanding ofevents that promulgate from the effector:target cell interface

Materials and Methods

Immunological and Chemical Reagents

mAbs to 1, 2, 3, 4, 5, 6, 1, 4, 5, _(L), LFA-1, Pyk2, SyK, Mac-1,ICAM-1, and ICAM-3 (PharMingen). CD3, CD4, CD8, CD19, CD56, CD45 directconjugates (FITC/PE/PERCP/APC/Biotin), granzyme-A-FITC (PharMingen).Perforin-CY5 and CD8-CY5PE (gift from the Herzenberg Laboratory,Stanford University). ICAM-2 mAb and ICAM-2-FITC (IC2/2 ResearchDiagnostics). Anti-phospho PYK2(Y402), anti-phospho-p44/42 (pT185Py187)(Biosource). Anti-phospho PKC/(Thr638), anti-phospho-Syk(Tyr525/526),anti-phosphoRaf1(Ser259) (Cell Signaling Technologies). Protein andchemical reagents used: fluorescein isothiocyanate (FITC) (Pierce),Alexa fluor dye series 488, 546, 568, 633, taxol-alexa546,phalloidin-alexa633, and CFSE (Molecular Probes). Tyrphostin A9 and 18,SB203580, piceatannol, bisindolylmaleimide I and II, herbimycin A(Calbiochem). Emodin, genistein, DMSO, PMA, PHA, staurosporine,ionomycin, propidium iodide, cytochalisin D (Sigma). Protein A/G agarose(SCBT). Recombinant human IL-2 (Roche), recombinant human ICAM-1-FC,ICAM2-FC, ICAM3-FC (Genzyme). Secondary antibodies to mouse and rabbitIgG (Santa Cruz). Mock treatments consisted of mouse IgG (forantibodies), 1% BSA (for proteins), or 0.001% DMSO vehicle (forchemicals).

Cell Culture

NIH3T3 cells were maintained in DMEM, 10% DCS, 1% PSQ (DuelbeccoModified Eagle Media, 10% Donor calf serum, 1% penicillin-streptomycin(1000 units/ml and 2 mM L-glutamine PSQ). Jurkat T-cells were maintainedin RPMI-1640, 10% FCS, 1% PSQ at 1×10⁵ cells/ml and serum starved 12hours for all functional assays. Cells were maintained at 5% CO₂/37° C.humidified incubator. Human peripheral blood monocytes were obtained byFicoll-plaque density centrifugation (Amersham Pharmacia, Uppsala,Sweden) of whole blood from healthy donors (Stanford Blood Bank) anddepleted for adherent cells. Magnetically activated cell sorting wasused to negatively isolate na□ve CD8⁺ T cells for studies as indicated(Dynal, Oslo, Norway).

Soluble ICAM-2 Generation and Synthesis of ICAM-2-FITC and ICAM2-Beads

Full length ICAM2 cDNA was obtained from Jurkat cells and cloned intoretroviral vector PBM-Z-IN at the BamHl/Sall site as described (Perez etal., 2002). Human ICAM-2 was overexpressed in NIH3T3 cells by retroviralinfection and harvested by immunoaffinity chromatography. ICAM-2 wasaffinity purified using a two step lysing procedure and subsequentpurification on an anti-ICAM-2 solid support. Cells were lysed in bufferA (20 mM Tris pH 7.5, 150 mM NaCl 1 mM EDTA 1 mM EGTA 0.1% NP40, 2.5 mMNa₂PO₄, 1 mM-glycerolphosphate, 1 mM Na₃VO₄, 1 ug/ml Leupeptin, 1 mMPMSF, protease inhibitor cocktail tablet (Boehringer Mannheim) for 5 min4° C., and subsequently permeabilized with 50% v/v with buffer B (BufferA plus 1% Triton-X-100) for 30 min 4° C. Supernatant was harvested bycentrifugation (14,000 RPM, 5 min, 4° C.). An Anti-ICAM-2 pAb to theC-terminal (4 mgs, Santa Cruz) was conjugated to an Affi-Gel Hzactivated support (Biorad) as suggested by manufacturer. This supportcouples Ig molecules via the FC region, resulting in higher antigenbinding capacity. Batch lysate of harvested supernatant was performed(4° C., for 2 hrs), and washed 4 times in buffer C (0.1% Tween™-20(polvsorbate 20), PBS pH 7.4). ICAM-2 protein was eluted by 4.5 M NaCI(in Tris pH 6.8), dialyzed overnight (in PBS pH 7.4, 0.001% azide, 0.01%glycerol, 4° C.), concentrated using size exclusion spin chromatographyand stabilized using 0.01% glycerol. Anti-ICAM-2 solid support wasre-equilibrated in buffer C, stored in 0.001% thimerosol and re-used upto 3 times. Purity was >98% as assessed by coomasie gel. Size exclusionchromatography removed higher molecular weight aggregates and were notobserved on purified ICAM-2 by native gel electrophoresis. 20 mgs werepurified by this method and used for this study. ICAM-2-FITC synthesiswas achieved by chemical conjugation to NHS-Fluorescein (Pierce) andunreactive dye was removed by gel filtration. ICAM-2-FITC probe did notintegrate into trypsinized Jurkat cells or bind when blocked by LFA-1antibody clones TS1/22 or TS1/18 (Developmental Hybridoma Studies Bank)or unlabeled ICAM-2 protein as determined by flow cytometry. ICAM-2-FITCbinding was not blocked by 2 integrin clone CT104 (Santa Cruz). PurifiedICAM-2 was comparable to human recombinant ICAM-2FC fusion proteinpurified from NSO murine myeloma cells (Genzyme). ICAM-1 FC and ICAM-3FCwere also purified from NSO cells (Genzyme). Proteins were spun at14,000 RPM, 5 min prior to use. 1 mg of ICAM-2 protein was conjugated to2X10⁸ epoxy activated beads as suggested by manufacturer (Dynal). 4X10⁵beads containing a total of 2 g ICAM-2 protein were used as indicated.Gel imaging was performed on a VersaDoc machine (Biorad) and analyzedusing Quantity One quantitation software (Biorad).

Flow Cytometry

Intracellular and extracellular staining was performed as described(Perez and Nolan, 2002). Intracellular probes for active kinases weremade by conjugating phospho-specific antibodies to the Alexa Fluor dyeseries as described and used in phospho-protein optimized conditions(Perez and Nolan, 2002). Kinetic analyses was performed by directapplication of fixation buffer in time synchronized 96-wells maintainedat 37° C. Intracellular actin and microtubule staining was performedusing phalloidin-Alexa633 and taxol-Alexa546 dyes (Molecular Probes).Adhesion and clustering assays were performed using ICAM-2-FITC asdescribed in text. LFA-1 activation was assessed by eithermAb24-Alexa633 or mAb24-Alexa546 conjugate, surface stained at 37° C.Flow cytometry data are representative of 3 independent experiments of10⁶ cells/sample. 10-50,000 events were collected and manuallycalibrated on a FACSCalibur machine. Data plotted in bar graph format isexpressed as geometric mean fluorescence intensity (MFI) and normalizedfor isotype controls. Log ratios are defined as the MFI of stimulus tothe MFI of unstimulated cells. Data was analyzed using Flowjo software(Treestar).

Single Cell ICAM-2 Binding Measurements

Percentage of ICAM-2-FITC binding was expressed as100*((MFI_(exp)−MFI_(ctl))/(MFI_(final)−MFI_(ctl))), where MFI_(exp)equals the mean fluorescent intensity of experimental concentration,MFI_(ctl) equals mean fluorescent intensity of unstained cells,MFI_(final) equals mean fluorescent intensity of final concentrationthat saturated binding. The samples were incubated with finalconcentrations as indicted in FIG. 3 from USSN 10/193,462 for 30 min at37° C. in 50 _L staining media (def RPMI, 4%FCS), washed 1X (500 μl, PBSpH 7.4, containing 1mM EDTA), and resuspended in 100 μl (1%paraformaldehyde). Dilution factor of staining conditions and molecularweight of 72.1 kD was used in determining molar concentrations. Thestaining buffer contained 2.4 mM calcium and 2 mM magnesium. The datawere fit to the equation V=V_(max)[S]/(K_(m)+[S]) where V is the percentbound, [S] is the ICAM-2-FITC concentration, and K, is theMichaelis-Menten binding constant using Kaleidagraph software.

Laser Scanning Confocal microscopy

Jurkat cells were treated as indicated and adhered to poly-L-lysine(Sigma) coated sterilized coverslips (1 mg/ml, 30 min) by mildcentrifugation (1000 RPM, 10 min), washed twice in phosphate bufferedsaline pH 7.4 (PBS) and fixed in 2.7% paraformaldehyde (in PBS). Cellswere permeabilized (5 min, 0.1% Triton-X-100 in PBS), washed twice inPBS, blocked in 4% FCS, and subjected to antibody or intracellularstaining as indicated. Stained coverslips were mounted and visualizedusing a Zeiss laser scanning confocal microscope 510.

Immunoprecipitations, Immunoblotting and Kinase Assays

Cell extracts were prepared by washing 2×10⁶ cells (treated asindicated) in ice cold PBS and harvesting in lysis buffer (20 mM Tris pH7.5, 150 mM NaCl 1 mM EDTA 1 mM EGTA, 1% Triton X-100, 2.5 mM Na₂PO₄, 1mM-glycerolphosphate, 1 mM Na₃VO₄ 1 g/ml Leupeptin, 1 mM PMSF, proteaseinhibitor cocktail tablet (Boehringer Mannheim). Extracts werecentrifuged 14,000 RPM (5 min, 4° C.) and 10-20 g (BCA protein assay(Pierce)) were immunoblotted using standard procedures.Immunoprecipitations (IP) were pre-cleared with protein A/G plus-agarosebeads, incubated with primary ab (1 h), protein A/G plus-agarose beads(1 h) and washed 4× with lysis buffer. Blots were incubated with theindicated antibodies and developed using ECL (Amersham). Immunoblotsstripped and reprobed (as indicated) were done by incubating withstripping buffer (62.5 mM Tris, pH 6.8, 10% SDS, 1%-mercaptoethanol) (30min, 55° C.). MAPK activity was detected by a p44/42 MAPK kinase kit assuggested by manufacturer (Cell Signaling Technologies).

Cytolytic Activity, Perforin Release Assays, and Conjugate FormationAssays

Target cell lysis was measured by flow cytometric based detection ofCFSE labeled HL60 cells. HL60 cells were labeled with 1 g of CFSE (30min, 37° C.). Targets were washed twice and mock treated, IL-2 activated(100 U/ml), CD3/CD28 activated (1 g/ml), or treated with ICAM2 beads orsoluble ICAM-1, -2, or 3 (10 g/ml, 30 min, 37° C.) before plating at 10⁴target cells/well of a 96-well round bottom plate. CTLs were added at50:1, 25:1, and 12.5:1 E:T ratio, and incubated at 37° C. for 4 hrs.Cells were then processed for multiparameter flow cytometry andintracellular perforin stain. Percent specific lysis was calculated bythe following equation: % specific lysis=100-100×(experimental HL60count/total control HL60 count). HL60 counts were detectable by the CFSEfluorescence. Percent perforin was calculated by the following equation:% perforin=100×[(experimental perforin MFI−isotype mAb MFI)/(totalperforin MFI−isotype mAb MFI)]. MFI refers to mean fluorescent intensityof flow cytoemtric based intracellular detection. Cell conjugates weredetermined by flow cytometry as described (Morgan et al., 2001).Chemical inhibition was done at 10 M of indicated compound (30 min, 37°C.) prior to stimulation as indicated. All experiments were performed intriplicate.

Example 2

Phospho-protein driven signaling networks support altered growth factorresponses of tumors and are considered crucial to initiation andmaintenance of tumor cell pathology. In acute myeloid leukemia (AML),dysregulated growth and inhibition of apoptosis lead to the accumulationof immature myeloid progenitor cells and oncogenic progression. We showhere that surveying phospho-protein responses to cytokine stimulation,in addition to widely studied basal phosphorylation states, revealsdysregulated signaling nodes and enables identification of a signalingpathology profile. We further show that unsupervised clustering ofphospho-protein profiles can be used to identify AML patient groups thatcorrelate with prognostic indicators, such as mutation of the receptortyrosine kinase Flt3 and chemotherapy resistance. In patients with Flt3mutations, the biochemical differences used to define patient groupsindicated a potentiated response of Stat3 and Stat5 to the myeloidcytokines GM-CSF and G-CSF.

In a potentiated model of signal transduction, dysregulated cell growthis predicted to be accompanied by sensitized differences in the internalsignaling states of cells. One key signal transduction network active incells that are considered progenitors of AML is the Janus kinase/SignalTransducers and Activators of Transcription (STAT) pathway. Statproteins are important in hematopoietic cytokine receptor signalingpathways that normally regulate cell proliferation, differentiation, andsurvival. In AML, several reports suggest that STATs, such as Stat3 andStat5, are involved in oncogenesis are constitutively activated andmight be effective targets for therapeutic intervention. A parallelsignaling system known to be constitutively activated in some AMLs isthe Ras and mitogen activated protein kinase (Ras-MAPK) pathway(involving the MAPK proteins p38 and Erk1/2). In addition to alteredbasal phosphorylation of key proteins, a potentiated model of signaltransduction predicts that cancer cells may have additional signalingchanges that cause them to react in an inappropriate or sensitizedmanner to environmental inputs. Exposure to such environmental inputscould reveal additional measures of difference in the phospho-proteinnetworks that are reflective—either causal or reactive—of the underlyingchanges required to initiate or sustain the growth of a particular tumorsubtype. Therefore, phospho-protein signaling variations among tumorsmight be employed as markers of differential disease course and responseto treatment.

We sought to test whether signaling could be used to classify pathologicdifferences in leukemic blasts from a well-characterized cohort of 30adult AML patients. To meet a standard of measuring multiple eventssimultaneously in a more native context we applied intracellularphospho-specific flow cytometry to detect multiple phosphorylated,activated signaling molecules in primary leukemic cells drawn from thesepatients. A cytokine response panel, composed of 36 phospho-proteinstates (6 basal states and 30 cytokine responses), was designed tosurvey altered signal transduction of tumor cells (FIG. 1 a). Eachsquare in the grid approximates a multidimensional flow cytometry filethat contained 30,000 cell events. The cytokine responses of eachphospho-protein node were compared to the basal state by calculating thelog 2 fold difference in median fluorescence intensity (MFI) ofstimulated samples divided by unstimulated. Although the data arevisually simplified in a response panel, the multiparameter flowcytometry data are available for a detailed inspection of single-cellphospho-protein responses of interesting subsets.

The cytokine response panel included detection of phosphorylated Stat1,Stat3, Stat5, Stat6, p38, and Erk1/2. These proteins provide a survey ofthe signaling networks that are suspected contributors toleukemogenesis. We collected data on unstimulated cells and cellsstimulated for 15 minutes with 20 ng/mL of Flt3 ligand (FL), GM-CSF,G-CSF, IL-3, or IFN-γ. As an example, we applied this panel to the U937histiocytic lymphoma cell line (FIG. 1 a), the HL-60 AML cell line andmultiple samples of a CD33+ subset from normal peripheral blood (FIG. 1b). CD33+ cells represent differentiated cells of myeloid lineage andwere used to assess variability in cytokine responses among samples fromblood donors. In general, the cytokine responses of normal lymphocytesubsets varied very little between donors (σ2<0.1, n=6) and signaling incell lines and CD33+ cells reflected previously reported mechanisms ofsignal transduction 4. We then prepared cytokine response grid for sixof the 30 AML patient samples (FIG. 1 b, AML-P01 through P06). Repeatmeasurements of cell lines, differentiated cell samples, and AML sampleswere collected (n≦3) and verified that the technique and monoclonalantibodies displayed a level of reproducibility similar to previousstudies. Importantly, however, the response to stimulation highlightedsimilarities and differences in signaling across patient tumor samples.For example, although AML-P02 and AML-P03 displayed many similarities,AML-P02 responded to GM-CSF and G-CSF through phosphorylation of Stat5while AML-P03 responded only to GM-CSF (FIG. 1 b). Additionally,although most patient samples and controls in this set displayed potentphosphorylation of Stat1 following IFN-g, AML-P05 and AML-P01 lackedthis response. We expanded this cytokine response panel to include all30 AML patients (FIG. 2 a) to search for significant similarities anddifferences among patient responses. Prior studies of AML oncogenesisfocused on constitutive signal transduction pathways and worked from thehypothesis that this activation level might be sufficient to monitor thedysregulation of cell signaling status Differences in basal STAT andRas-MAPK protein phosphorylation were consistent with previous reportsshowing widespread, varying constitutive activity of signaling pathwaysin AML.

However, in support of the hypothesis that differences in underlyingtumour mutations might lead to difference in potentiated signaling,phosphoprotein responses in many of the primary AML blasts showedconsiderable induction and variance of STAT and RAS/MAP-K pathway memberphosphorylation (FIGS. 2 a,b). In addition to the six basalphosphorylation states, we identified seven of the thirty cytokineresponse states that displayed significant variance across AML patientsamples (σ2>0.1, FIG. 2 b, highlighted in yellow). These were: (i)phosphorylation of Stat3 following G-CSF, (ii-v) phosphorylation ofStat5 following GM-CSF, G-CSF, IL-3, and IFN-γ, (vi) Stat1phosphorylation following IFN-γ, and (vii) phosphorylation of Erk1/2following FL. Remaining tumor cytokine responses displayed variation ator less than the variance in normal, differentiated CD33+ cells and thetwo closest node states to the significance threshold did not affectsubsequent patient groupings. A graph of the absolute median against thevariance indicates the signal to noise threshold and the relationship ofvariance to the median basal state or cytokine response (FIG. 2 c).

Based on the potentiated signaling hypothesis, we predicted that these13 phospho-protein states represented underlying oncogenic eventsrequired to initiate or sustain differential tumor pathology and couldbe used to stratify patient risk. We composed a signal transductionbased classification for AML using unsupervised clustering of thesephospho-protein biosignatures. When patients are clustered according toa biological hypothesis and then subsequently tested for correlation toclinical parameters—as opposed to pre-grouping patients by clinicaloutcome and then testing each node state as a hypotheses—the statisticalperils of multiple t-tests can be avoided. This method also allows fordisease subgroups to be identified even when normal control cells arerare or difficult to obtain in bulk, as is the case with myeloidprogenitor cells, because differences among experimental samples areused to define groups rather than comparison of each sample to aproposed normal control.

To group biosignatures by similarity (FIG. 3 a) we employed the completelinkage hierarchical clustering algorithm available in MultipleExperiment Viewer (MeV)http(colon)//www(dot)tiqr(dot)orq/software/tm4/mev(dot)html). Similaritygrouping identified four main clusters of AML patients (FIG. 3 b). Eachof these was termed a Signaling Cluster (SC) and was referred to basedon the primary signaling profile of the cluster: (i) SC-NP displayedlittle or no potentiated cytokine responses and had varied basalphosphorylation (underlined letters denote the cognate acronym), (ii)SC-PB displayed both potentiated cytokine responses and high basalphospho-protein states, (iii) SC-P1 displayed intense potentiatedsignaling, low basal phosphorylation, and a p-Stat1 response to IFN-y,and (iv) SC-P2 displayed many potentiated cytokine responses in thecontext of low basal phosphorylation.

To test the potentiated signaling hypothesis we asked whether clinicalfactors previously determined to be prognostic for AML correlatedsignificantly with the biosignature-derived SCs (FIG. 3 b). Resistanceto course 1 chemotherapy—cytarabine plus an anthracycline given topatients healthy enough to withstand associated cytotoxicity—correlatedsignificantly with SC-P2 (p=0.002, FIG. 3 b). Patients in SC-P2constituted 33% of this cohort (10/30). This finding demonstrates thatsignal transduction based classification of human cancer, a priori ofknowledge of clinical outcome, can produce a patient classification thatis predictive of response to therapy.

One genetic change we predicted might potentiate pre-existing myeloidsignaling networks was mutation of the Flt3 receptor tyrosine kinase.Abnormalities of Flt3 are detected in approximately 30% of AML patientsand are well-established as a negative prognostic indicator in AML11,12.

Experiments in cell lines have suggested that expression of mutant Flt3gives rise to constitutive Stat5 and Ras-MAPK activity, however thisconnection was not observed in previously published comparisons of basalStat5 phosphorylation in Flt3 mutant and wild type patient-derivedprimary AML blasts27.

Notably, the AML patient samples that displayed high myeloid cytokineresponses contained internal tandem duplication of Flt3—whereas thesamples with a non-potentiated SC profile lacked these mutations(p=0.02). Of patients with Flt3 mutations that do not cluster in SC-P2,4/7 showed other mutations including either Asp835 Flt3 mutation orN-Ras mutations (data not shown). As reported by others, we observed nosignificant difference in the basal phosphorylation of Stat5 in eithernormal or Flt3-mutant AML patients (FIG. 4 a).

Potentiated signaling of Stat5 and, to a lesser extent, Stat3 was commonin the AML patient groups displaying Flt3 mutations. Phosphorylation ofStat5 following GM-CSF and G-CSF was significantly potentiated inpatients with Flt3 mutations (p=0.04 and 0.02, respectively, FIG. 4 aand FIG. 4 b). It is notable that Stat3 phosphorylation following G-CSF(which was the only Stat3 biosignature state displaying significantvariance among AMLs) also correlated with Flt3 mutation (p=0.01, FIG. 4a and FIG. 4 b). To represent the effect of Flt3 mutation on myeloidsignal transduction nodes we summed the four cytokine response nodestates (p-Stat3 following G-CSF, and p-Stat5 following GM-CSF, G-CSF,and IL-3) and graphed the correlation between the myeloid cytokineresponse and Flt3 mutation in AML patient samples. We observe a strikingcorrelation that strongly support the notion that Flt3 mutationspotentiate signaling through pre-existing potentiated pathways in AML(p=0.005, FIG. 4 a). This might indicate patients displaying potentiatedmyeloid signal transduction, and especially those with the profile ofSC-P2, might respond well to therapies involving STAT pathwayinhibitors.

Additionally, these data suggest that each SC grouping reflectsproliferative advantages required for leukemogenesis that, when combinedwith other genomic and proteomic changes, produced common signatures inthe mitogenic signaling network of leukemic cells. Correlation of sixcell surface antigens with SC groups or Flt3 identified the unexpectedloss of the CD15/Lewis X as significantly correlated with SC-P2 and Flt3mutation (χ2 test indicated p=0.03 and p<0.001, respectively, FIG. 3 b).

The profile of SC-NP, which included 36.7% of patients (11/30), wascomprised primarily by low to no cytokine response and lowphosphorylation of Stat1 following IFN-γ (FIG. 3 a). Additionalresponses associated with this group were low Stat5 phosphorylationfollowing IL-3 and low Stat3 phosphorylation following G-CSF, both ofwhich are the opposite of changes observed in SC-P2. Interestingly, inSC-NP there were cytogenetic alterations 10 that fell into a closelyrelated branch (FIG. 3 a, hierarchy branch AML-P23 through AML-P30)containing two patients with a 9; 11 translocation (AML-P27 and AML-P24)and two patients with a loss of chromosome 5 (AML-P23 and AML-P17).Patients with no detectable cytogenetic alterations formed a closelyrelated branch of SC-P1 (FIG. 3 a, hierarchy branch AML-P13 throughAML-P20). The division of altered or diploid cytogenetics among branchesof SC-NP and SC-P1 was statistically significant (p<0.001). The onlyother patient with a 9; 11 translocation, AML-P03, contained an Asp835mutation of Flt3 and was profiled as SC-PB. AML-P26, which displayed anSC-NP profile, was the sole patient in this cohort where a copy ofchromosome 17, the locus of Stat5 and Stat3, had been lost.

A critical advantage of single cell based biosignatures is that, asshown above, they approximate complex data well enough to allowbiologically significant clustering while retaining the underlying flowdata for further examination. In addition to validating controls andcomparing the magnitude of responses, we examined the original flowcytometry histograms and 2D plots and found that higher dimensionalrepresentations of the data identified subsets of cells with uniquefeatures. For instance, the 2D contour plots for the GM-CSF and G-CSFresponse of Stat5 and Stat3 are shown grouped by Flt3 mutational statusfor 10 representative AML patient samples (FIG. 4 b). Note that althoughStat3 and Stat5 are both thought to be constitutively active in leukemiaand share many structural similarities, Flt3 mutation correlated withpotentiated Stat5 phosphorylation following GM-CSF but had norelationship to Stat3 phosphorylation (top row). In contrast, in mostcases Flt3 mutation appeared to correlate with potentiation of both thep-Stat5/G-CSF node and the p-Stat3/G-CSF node in individual primary AMLblast cells (bottom row). This suggests strongly that Flt3 mutation can,in significant subset(s) of cells as observed in two dimensional flowcytometry, lead to unexpected co-activation of important phospho-proteintranscription factors.

Phospho-protein signaling of blast cells from AML-P05 were classified byMeV software as having a profile similar to SC-NP despite the detectionof Flt3 mutation. The presence of a defined subset of AML-P05 blastcells with p-Stat5 and p-Stat3 potentiation similar to the profile ofSC-P2 was surprising and suggests that some cells may have acquiredadditional mutations leading to altered signaling (FIG. 4 b). AML-P05,like all other patients with Flt3-ITD and a non-SC-P2 profile, went intoremission following course 1 chemotherapy (p<0.001, FIG. 3 b). Inaddition, the well recognized limitation to classification of tumourmicro-heterogeneity might be overcome using multi-parameter approachesthat distinguish tumor cell subset responses to input stimuli.Extraction of high dimensional information could be applied to furtherrefine our understanding of systems such as that presented here,especially when additional phospho-proteins or other single cell eventsare measured simultaneously. Whether these subset differences representsimply non-responsive cells, cancer ‘stem’ cells2, or other stages ofclonal evolution within the tumour remains to be determined.

The mechanism-based classification of AML reported here indicates thatthe phospho-proteome can be informative of tumor pathology. In addition,the cytokine response approach demonstrated could readily be applied tofields outside cancer research. By provoking cell populations to respondto external stimuli, one can distinguish differences in underlyingsignaling pathologies and thereby classify patient populations. In thisregard it was striking that AML blasts responded to cytokine stimulationas they are commonly thought of as having constitutively active STAT andRas-MAPK signaling. The finding that potentiated cytokine responses ofthese tumors can be combined with information on basal phosphorylationstates and clustered to create a classification with clinical relevancewill be important for clinical groups designing and testing mechanismbased cancer therapies. The results suggest that patient chemotherapyregimes, including kinase inhibitors or other therapeutics, might betailored for and directed to specific cell subsets withinbiosignature-defined patient response groups.

Materials and Methods

Patients and Preparation of AML Blasts.

The study was approved by the local Ethics Committee and samplescollected after informed consent. Samples were selected from a largegroup of consecutive patients with de novo AML and high peripheral bloodblast counts20. These patients were admitted to the hospital from April1999 to August 2003, the median age was 60 and ages ranged from 29 to84. As these patients were selected for high blast counts, enriched AMLcell populations were prepared using a simple density gradientseparation of peripheral blood samples (Ficoll-Hypaque; NyCoMed, Oslo,Norway; specific density 1.077) before safe, standardizedcryopreservation according to previously developed techniques28. Thesepatients represent the latter portion of a group studied previously forFlt3 signaling and mutation in AML20. The acute myeloid leukemia cellline HL-60 and the monocytoid lymphoma cell line U-937 was from AmericanType Culture Collection www(dot)atcc(dot)orq) and cultured in RPMImedium (Invitrogen, Carlsbad, Calif., USA)+10% Fetal Calf Serum(HyClone, South Logan, Utah, USA).

Stimulation of AML Blasts.

Peripheral blood containing >95% AML blasts was thawed into 5 mL StemSpan H3000 defined, serum free medium (Stem Cell Technologies,Vancouver, BC, Canada), counted, and resuspended at 2×106 cells per mL.Six FACS tubes (Falcon 2052, BD-Biosciences, San Jose, Calif.) were thenfilled with 2 mL of each leukemia sample and allowed to rest at 37° C.for 2 h. AML blasts were resuspended gently to prevent aggregation andallowed to rest at 37° C. for another 45 minutes. At this time vehicle(deficient RPMI medium, Invitrogen, Carlsbad, Calif.) or 40 μL ofstimulus was added to each tube to a final concentration of 20 ng/mL.Stimuli included human recombinant Flt3 ligand (FL), GM-CSF, G-CSF,IL-3, and IFN-γ (all cytokines from Peprotech, Inc., Rocky Hill, N.J.,USA). Samples were returned to the 37° C. incubator for 15 minutes toallow signal transduction and phosphorylation, after which 100 μL of 32%para-formaldehyde (PFA, Electron Microscopy Services Fort Washington,Pa., USA) was added to each 2 mL tube of cells to a final concentrationof 1.6%. Cells were fixed for 15 minutes at room temperature,permeabilized by resuspension in 2 mL ice cold methanol for 10 minutes,and stored at 4° C. until being stained for flow cytometry.

Intracellular Phospho-Specific Flow Cytometry.

PFA fixed, methanol permeabilized AML blasts were rehydrated by adding 2mL phosphate buffered saline (PBS), gentle resuspension, and thencentrifugation. The cell pellet was washed once with 2 mL PBS,resuspended in 150 μL PBS+0.1% BSA (Sigma, St. Louis, Mo., USA), andsplit evenly into three new FACS tubes. 50 μL of an antibody mixcontaining 0.065 μg primary conjugated phospho-specific antibody persample was added to each tube of AML blasts and staining proceeded for20 minutes at room temperature. Alexa (Ax) dye (Molecular Probes,Eugene, Oreg., USA) coupled primary conjugated antibodies (all fromBD-Pharmingen, San Diego, Calif., USA) included antibodies againstphospho-Stat3(Y705)-Ax488, phospho-Stat5(Y694)-Ax647,phospho-Stat6(Y641)-Ax488, phospho-Stat1 (Y701)-Ax647,phospho-p38(T1801Y182)-Ax488, and phospho-Erk1/2(T202/Y204)-Ax647 andwere applied in pairs (Stat5/Stat3, Stat1/Stat6, and Erk/p38) forsubsequent detection of Alexa-488 and Alexa 647 (Molecular Probes,Eugene, Oreg.) on FL-1 and FL-4, respectively. Stained AML blasts werethen washed by adding 2 mL PBS+0.1% BSA and resuspended in a finalvolume of 200 μL PBS. Approximately 30,000 ungated events were collectedfor each sample on a benchtop FACSCalibur dual-laser cytometer (BectonDickinson, Franklin Lakes, N.J., USA). When employed to detect cells ofmyeloid lineage in normal PBL, antibodies against CD33 werephycoerythrin conjugated (clone WM53, BD-Pharmingen, San Diego, Calif.,USA) and detected on FL-2.

Statistical Analysis.

Changes in phosphorylation of STAT and Ras-MAPK proteins followingcytokine stimulation were approximated by calculating the log₂ foldmedian fluorescence index (MFI) of stimulated over unstimulated cellpopulations. Differences in basal phosphorylation were compared bycalculating the log2 fold MFI of a sample over the minimum among tumors.To determine the statistical significance of observed versus expecteddistributions we used a χ² test (FIG. 3 b), and to determine thesignificance of the difference in mean of two populations we used astudent's t-test (α=0.05, FIG. 4 a). Standard t-tests were used tovalidate existing hypotheses.

Example 3

The following example employs the methods and compositions of theinstant invention to generate and analyze potentiation data based onexposure to a specific drug.

Initial patient samples, including five collections from each of twelvepatients (total 60 samples), are aliquoted into 96-well plates toaccommodate the cytokine responses panel shown in Table 1. Each 96 wellplate then be stained with either the 4-color or 6-color antibodycocktails detailed in Table 2.

In addition to the patient samples, 3 cell lines (U937, HL60, MV411) areemployed for in vitro drug investigation. The cell lines areindividually treated with drug at 5 time points (1 hr, 2, hr, 4 hr, 12hr, 24 hr) prior to cytokine stimulation. The resulting 15 samples arealiquoted into 96-well plates and stained as described above.

Finally, collections from six normal blood donors are processed as“control” samples. These control samples include five collections fromeach of the six donors and are processed identically to the initialpatient samples. In addition, five collections from each of the sixdonors are processed identically as the three cell lines employed in thein vitro drug investigation.

Flow cytometric data is collected for each of the fully processedsamples and analyzed via bioinformatics tools described above andstatistical significance analysis is generated based in the processedflow data.

TABLE 1 Generalized protocol for study of potentiation. 1) Cells aresuspended in a small volume of serum free H3000 stem span media. 2)Cells are counted. A minimum of 20 million viable cells from each shouldbe present in the sample (for four core stains), plus 5 million for eachstored sample. 3) Cells are diluted to 6.2 × 10{circumflex over ( )}5cells/100 uL, and 100 uL of each is aliquoted into a 96-well plate, asshown below (Experimental Layout). 4) Stimulations are added at 10 ng/mLinto the colums of cells. Stimulation progresses for 15 minutes at 37°C. 5) To end stimulation, cells are fixed by adding 10 uL 32% PFA toeach well (5 min at room temperature). 6) Cells are pelleted andresuspended in methanol to permeabilize. Cells can be stored at thispoint for 3-6 weeks, or stained with labeled antibodies and analyzedright away. 7) The response panel is approximated in a two dimensionalarray of nodes (in rows) and states (in columns), similar to theexperimental layout. Typically, the log₂ of [MFI stimulated/MFIunstimulated] is taken. For basal nodes, the log₂ of [MFI basal x/MFItumor sample minimum basal] can be used to place measurements on thesame scale. 8) To identify a biosignature, the node states for all tumorsamples are collected and the variance across tumors studied. Formultiple drug timepoints, each timepoint of a sample is treated as aseparate sample. Those node states with variance greater than that seenin a normal cell are typically included in the biosignature. 9)Unsupervised clustering of biosignature node states is used to groupsamples. 10) Distribution of clinical parameters in resulting groups isdetermined using statistical significance testing (Chi Squared andStudent's t-test on previously defined hypotheses). 11) Groups ofpatient samples with similar potentiation are given designations. 12)Pathway maps, based on all possible pathway interactions observed, arehighlighted and dimmed according to observations of each group. Ingeneral, if more than half the samples in the group display potentiationabove the median for all samples, then that pathway is highlighted.Otherwise, the pathway is dimmed. 13) Pathway maps can be built out forindividual patients based on their observed potentiation, and based onthese maps a patient sample can be phenotyped as belonging to a group(and will be predicted to have similar clinical outcome as that group).14) A predictive panel for the disease is made, composed of biosignaturenode states, and validated on an additional set of patient samplessimilar to the original. This panel is minimized and optimized to be asaccurate as possible with as few as possible node state measurements fora given sample.

TABLE 2 96 well experimental response panel layout for potentiationexample using one sample (corresponding to a patient sample taken at adrug treatment timepoint).

Colored rows will be stained for analysis, grey rows, cell numberspermitting, could be banked in methanol for future study. Banked samplescould be used for genetic analysis, staining other targets, orrepeat/control stainings. Designations a1-g7 refer to 96 well platecoordinates.

TABLE 3 4-color and 6-color Stains Stain 1 Stain 2 Stain 3 Stain 44-color pstat1(Y701)-AX488 pstat5(Y694)-AX488 p-flt3-AX488 p-Flt3-AX488pstat3(Y705)-pe p-erk1/2-PE pstat4(Y693)-AX546 p-erk1/2-PEpstat6(Y694)-AX647 p-p38-AX647 pstat3(S727)-AX647 pstat5(Y694)-AX647CD33-percpcy5.5 CD33-percpcy5.5 CD33-percpcy5.5 CD33-percpcy5.5 6-colorpstat1(Y701)-AX488 pstat5(Y694)-AX488 p-Flt3-AX488 p-Flt3-AX488pstat3(Y705)-pe p-erk1/2-PE pstat4(Y693)-AX546 p-erk1/2-PEpstat6(Y694)-AX647 p-p38-AX647 pstat3(S727)-AX647 pstat5(Y694)-AX647CD33-percpcy5.5 CD33-percpcy5.5 CD33-percpcy5.5 CD33-percpcy5.5CD38-PeCy7 CD38-PeCy7 CD38-PeCy7 CD38-PeCy7 CD14-APCCy7 CD14-APCCy7CD14-APCCy7 CD14-APCCy7 TRUCOUNT BEADS TRUCOUNT BEADS TRUCOUNT BEADSTRUCOUNT BEADS

1. A method of detecting the activation state of at least a first and asecond activatable protein in single cells, said method comprising thesteps of: a) contacting a population of cells comprising a firstactivatable protein having a first and second isoform and a secondactivatable protein having a first and second isoform with a pluralityof activation state-specific binding elements, wherein said plurality ofactivation state-specific binding elements comprise: i) a firstactivation state-specific binding element that binds to said firstisoform of said first activatable protein; and ii) a second activationstate-specific binding element that binds to said first isoform of saidsecond activatable protein; wherein said first activation state-specificbinding element and said second activation state-specific bindingelement are distinguishably detectable, and b) detecting the presence orabsence of binding of said first and second binding elements in singlecells in said population of cells to determine the activation state ofsaid first and said second activatable proteins in said single cells. 2.The method according to claim 1, wherein said first binding element islabeled with a first label, said second binding element is labeled witha second label, and said first and second labels are distinguishablydetectable.
 3. The method according to claim 1, wherein said populationof cells are primary cells.
 4. The method according to claim 1, furthercomprising using the determined activation state of said first andsecond activatable proteins to determine the cellular status of at leastone cell.
 5. The method according to claim 4 wherein said cellularstatus is drug susceptibility.
 6. The method according to claim 1further comprising contacting said population with a candidate agentprior to said contacting with said binding elements.
 7. The methodaccording to claim 6 wherein said candidate agent is a potentiator. 8.The method according to claim 7 wherein said potentiator is a cytokine.9. The method according to claim 7 wherein said potentiator is a growthfactor.
 10. The method according to claim 1 wherein said activationstate is a phosphorylation state.
 11. The method according to claim 1wherein at least one of said activatable proteins is a kinase.
 12. Themethod according to claim 1 wherein at least one of said activatableproteins is a caspase.
 13. The method according to claim 1, wherein saiddetecting is performed with a detector selected from the groupconsisting of: flow cytometer; microscope having multiple channels offluorescence; plate reader configured to detect fluorescence,ultraviolet light, visible light, fluorescence resonance energy transfer(FRET), luminescence, quenching, two-photon excitation, and/or intensityredistribution; a charge-coupled device (CCD) camera configured tocapture and transform data and images into quantifiable formats; highperformance liquid chromatography (HPLC); mass spectrometer; andradioisotope detector.
 14. The method of claim 1, wherein the firstactivation state-specific binding element is an antibody.
 15. The methodof claim 14, wherein the antibody is selected from the group consistingof: anti-PI3K, anti-PIP2, anti-PIP3, anti-AKT-phospho-Ser473 (monoclonalantibody 4E2), anti-phospho AKT (Thr308), anti-p44/42 MAPK(anti-phospho-Thr202/Tyr204, anti-phospho Thr185/Tyr187), anti-phosphoTYK2 (Tyr1054/1055), anti-phospho p38 MAPK (Thr180/Tyr182: monoclonalantibody 28B10), anti-phospho JNK/SAPK (Thr183/Tyr185: monoclonalantibody G9), anti-phospho-tyrosine (P-tyr-100), anti-phospho-threonine,anti-phospho-PKC-PAN substrate antibody, anti-phospho-PKA-substrate,anti-phospho PYK2 (Tyr402), anti-phospho PKC (phospho Thr638),anti-phospho-Syk (Tyr525/526), anti-phospho Raf1 (Ser259),anti-phospho-stat1 (Tyr701), anti-phospho-Stat3 (Tyr705),anti-phospho-Stat5 (Tyr694), anti-phospho-Stat6 (Tyr641),anti-phospho-p38 (Thr180/Tyr182), and anti-phospho-Erk1/2(Thr202/Tyr204).
 16. The method of claim 1, wherein the secondactivation state-specific binding element is an antibody.
 17. The methodof claim 16, wherein the antibody is selected from the group consistingof: anti-PI3K, anti-PIP2, anti-PIPS, anti-AKT-phospho-Ser473 (monoclonalantibody 4E2), anti-phospho AKT (Thr308), anti-p44/42 MAPK(anti-phospho-Thr202/Tyr204, anti-phospho Thr185/Tyr187), anti-phosphoTYK2 (Tyr1054/1055), anti-phospho p38 MAPK (Thr180/Tyr182: monoclonalantibody 28B10), anti-phospho JNK/SAPK (Thr183/Tyr185: monoclonalantibody G9), anti-phospho-tyrosine (P-tyr-100), anti-phospho-threonine,anti-phospho-PKC-PAN substrate antibody, anti-phospho-PKA-substrate,anti-phospho PYK2 (Tyr402), anti-phospho PKC (phospho Thr638),anti-phospho-Syk (Tyr525/526), anti-phospho Raf1 (Ser259),anti-phospho-stat1 (Tyr701), anti-phospho-Stat3 (Tyr705),anti-phospho-Stat5 (Tyr694), anti-phospho-Stat6 (Tyr641),anti-phospho-p38 (Thr180/Tyr182), and anti-phospho-Erk1/2(Thr202/Tyr204).
 18. The method according to claim 1, wherein saidstate-specific binding element is indirectly labeled with a detectablesecondary binding moiety.
 19. The method according to claim 18, whereinsaid secondary binding moiety is selected from the group consisting of:antibody, biotin, avidin, streptavidin, member of a receptor/ligandpair, member of a carbohydrate/binding partner pair, and member of anucleic acid/nucleic acid binding protein pair.